David Dubois

Stella Fernandes

Souad Amiar

Sheena Dass
email: sheena.dass@univ-grenoble-alpes.fr

Nicholas J Katris

Cyrille Y Botté
email: cyrille.botte@univ-grenoble-alpes.fr

Yoshiki Yamaryo-Botté

Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages

Keywords: Toxoplasma gondii, fatty acid synthesis and elongation, apicoplast, membrane biogenesis, lipidomics, stable isotope labelling Tubulin, dinitroanilines, plant cells, Toxoplasma gondii, Plasmodium falciparum, virtual screening, small molecules, cell-based assays

Apicomplexan parasites are responsible for major human infectious diseases such as malaria and toxoplasmosis against which there are no efficient vaccines and rapid emergence of drug resistance. The propagation and survival of these parasites depends on complex metabolic interactions with their human host cells. Lipid synthesis is one such key pathway crucial for parasite survival, which relies on an essential combination or 'patchwork' of fatty acids synthesized de novo and scavenged from the host. Additionally, these parasites can modulate their metabolic capacities depending on the different hosts and their nutritional environments.

Availability of the appropriate amount of fatty acids is an essential determinant for successful adaptation of the parasite to various host cells. Since, fatty acid uptake is as essential as de novo fatty acid synthesis for the parasite growth and pathogenesis, therefore these fatty acid homeostatic pathways hold promise for specific drug targets. The molecular mechanism by which parasites combine and regulate the fatty acid flux through effective remodelling of their lipid metabolism, remains unknown. Therefore, to determine the same my PhD project investigated pivotal role of two families of enzymes in parasite fatty acid metabolism: acyl CoA synthetases (ACS) putatively allowing the activation of fatty acids and phosphatidic acid phosphatases (LIPIN) the gatekeepers for the synthesis of central lipid precursors. Phosphatidic acid (PA), the simplest glycerophospholipid, acts as the rate limiting central lipid molecule in this process of fatty acid flux-based homeostasis. During my PhD, I investigated the role of a Toxoplasma phosphatidic acid phosphatase called TgLIPIN, in metabolising the critical levels of PA through controlled channelling of parasite vs host derived fatty acids.

Toxoplasma tachyzoites are unable to survive a knockdown of LIPIN due to severe replication defect amongst several other membrane anomalies. This cytosolic enzyme catalyses the synthesis of diacylglycerol from phosphatidic acid, a central lipid precursor and signal transducer, as confirmed by heterologous complementation and lipidomics approaches. The disruption of TgLIPIN induces loss of parasite lipid storage concomitantly with an increase of the total lipid content (fatty acids), leading to rapid parasite death by 'lipotoxicity'. With the help of novel 13 C glucose-based fluxometrics approaches using GC-MS, we identified that the enzyme mainly use and controls the use host fatty acids (esp. C18:1), which become a lipotoxic source for the parasite when disrupting TgLIPIN. One of the physiological consequences is a toxic increase of lipids and free fatty acids killing the parasite. On the other hand, the downregulation of TgLIPIN induced a significant reduction of the apicoplast FASII activity indicating its activity regulates the parasite

I would like to thank my friends and my constant supporters Pratima and Vrushali. It is their unconditional love and patience that helped me ride through this adventurous journey. A special thanks to Keerthi, Dayana and Georgious for all the food binging and fun times. Thank you for your support during both happy and sad times. This acknowledgement would be incomplete without mentioning my grandfather who showed faith in me and supported me to achieve my goals in life. Finally, I would like to thank my family, my loving parents and brother. Despite being continents apart, their unfailing support, encouragement and love helped me sail through this incredible journey of 3 years.

de novo synthetic capacities in response to its scavenging activity. Electron microscopy reveals that TgLIPIN indirectly regulates nuclear membrane morphology and also the inner membrane complex biogenesis, suggesting the importance of lipid balance during the growth of parasite.

Overall, we suggest that TgLIPIN protects parasite against FA-induced toxicity allowing a normal replication cycle within its host by regulating the critical levels of phosphatidic acid.

The second project during my PhD focused on the enzymatic pathway involved in the activation of fatty acids within these parasites. In eukaryotes, the activation of fatty acids to their acyl-coA thioesters is a two-step energy requiring biochemical process that involves key enzymes called Acyl-coA synthetases (ACS). Since, fatty acids are virtually involved all aspects of cellular biochemistry, their activation pathway could be an Achilles heel for the parasite survival within its host. In an attempt to the same, we have identified a novel family of 7 enzymes in Toxoplasma gondii as putative acyl-coA synthases (TgACSs). All the putative TgACSs, tagged endogenously, localize to non-overlapping sub-cellular compartments of the parasite. Using parasite genetics approaches and GC-MS based lipidomics, TgACS3 was functionally characterised. Genetic ablation of TgACS3 affected parasite replication within its host. TgACS3 depletion reduced the overall fatty acid content within the parasite phospholipids alongside a significant increase in the amount of free fatty acids, further confirming its function as an ACS.

Another major question I addressed in my PhD (including collaborative papers), is the effect of host environmental/nutritional conditions affecting the adaptation of de novo FA synthesis and host FA scavenging capacities of the parasite. Growing these parasites in the presence of growth media supplemented with different amounts of FBS (Fetal bovine serum) mimics different host nutritional environments in vitro. Normally, high nutritional environment (10% FBS) was able to sustain better parasite growth in comparison to low nutritional status, in a concentration dependent manner (10% FBS>1% FBS> 0% FBS). FBS starvation significantly increased the presence of nile red stained lipid droplets within the parasite or the PV in a dose-dependent manner, suggesting adaptive metabolic capacity of the parasite. However, in case of parasites lacking TgLIPIN and TgACS3, the consequent growth defect was aggravated in the presence of high nutritional environment i.e. 10% FBS supplemented growth media in comparison to low nutrient conditions (1% and 0% FBS). We determined the reason of this opposite trend (parasite growth rate: 10%FBS<1%FBS<0%FBS) as the increase in the amount of free fatty acids which when supplemented with external rich source resulted in growth defect due to absence of pivotal enzymes involved in FA metabolism. This thesis presents data that confirm that the intracellular replication and survival of apicomplexan parasites is reliant on effective remodelling of their lipid metabolic capacity by FA flux derived de novo, from the host and its nutritional environment.

Les parasites apicomplexes sont des pathogènes unicellulaires eucaryotes responsables de maladies infectieuses humaines majeures de l'homme telles que le paludisme et la toxoplasmose.

Il n'existe à ce jour pas de vaccin efficace contre ces agents pathogènes. De plus, l'émergence des souches parasitaires résistantes aux traitements actuels, tous deux pointent l'urgence actuelle sur l'identification de nouvelles cibles thérapeutiques. La propagation et la survie de ces parasites dépendent des interactions métaboliques complexes des parasites avec leurs cellules hôtes humaines. La synthèse des lipides est une de ces voies clés cruciale pour la survie du parasite, qui repose sur une combinaison essentielle ou «patchwork» d'acides gras synthétisés de novo et récupérés de l'hôte. De plus, ces parasites peuvent moduler leurs capacités métaboliques en fonction des différents hôtes et de leurs environnements nutritionnels. La disponibilité de la quantité appropriée d'acides gras est un déterminant essentiel pour une adaptation réussie du parasite à diverses cellules hôtes. Étant donné que l'absorption des acides gras est aussi essentielle que la synthèse de novo des acides gras pour la croissance et la pathogenèse des parasites, ces voies permettant le transport et l'homéostasie des acides gras représentent donc des cibles thérapeutiques. Le mécanisme moléculaire par lequel les parasites combinent les acides gras issus des deux voies d'acquisition/synthèse et régulent le flux d'acides gras grâce à un remodelage efficace de leur métabolisme lipidique reste inconnu. Mon projet de doctorat a focalisé sur l'étude du rôle supposé pivot et essentiel de deux familles d'enzymes dans le métabolisme des acides gras parasitaires : les acyl synthétases d'acyl (ACS) permettant l'activation des acides gras et les acide phosphatidique phosphatases, (PAP/LIPIN) à l'origine de la synthèse des précurseurs centraux de tous les lipides majeurs du parasite. L'acide phosphatidique (PA) est le glycérophospholipide le plus simple structurellement. De plus, il est ke précurseur unique pour la synthèse de novo de toutes les classes de glycérophospholipides.

Le PA agit comme une classe lipidique centrale dont la synthèse limite la vitesse dans ce processus de flux et d'homéostasie des acides gras. Au cours de ma thèse, j'ai étudié le rôle d'une acide phosphatidique phosphatase (PAP) appelée TgLIPIN. TgLipin régule les niveaux critiques du PA cellulaires une canalisation contrôlée des acides gras issus soit de la synthèse de novo du parasite soit obtenus par vol des ressources de l'hôte. L'arrêt de l'expression de TgLIPIN entraine la mort intracellulaire du parasite par arrêt de la division du parasite et par d'importantes anomalies membranaires. Cette enzyme cytosolique catalyse la synthèse du diacylglycérol à partir de l'acide phosphatidique, un précurseur lipidique central et un transducteur de signal, comme confirmé par les approches de complémentation hétérologue et d'analyses lipidomiques. La perturbation de TgLIPIN induit une perte des classes des lipides de stockage parasitaires en même temps qu'une augmentation de la teneur totale en phosphoglycérolipides et des acides gras libres, entraînant une mort rapide des parasites par «lipotoxicité». À l'aide de fluxométrie par marquage au 13 C-glucose suivi d'analyse lipidomique en GC-MS, nous avons identifié que la source lipotoxique de ces acides gras toxiques est directement issu l'hôte (en particulier l'acide oléique, C18 :1). D'autre part, l'activité de la voie FASII est fortement réduite lors de la déplétion de TgLIPIN, suggérant que son activité contrôle aussi directement ou indirectement les voies de néosynthèse des acides gras. La microscopie électronique révèle que TgLIPIN régule indirectement la morphologie de la membrane nucléaire et également la biogenèse du complexe membrane interne (IMC), suggérant l'importance de l'équilibre lipidique pendant la croissance du parasite. Nos approches en fluxomique et lipidomique révèlent donc que TgLIPIN régule la balance synthétique du PA et du DAG, controlant ainsi soit la biogénèse membranaire active du parasite soit le stockage des lipides, respectivement. Mes travaux montrent donc que TgLipin est le régulateur métabolique central controllant la synthèse lipidique du parasite et permettant donc sa division intracellulaire controllée et sa survie intracellulaire.

Le second volet de monprojet de ma thèse a porté sur la voie enzymatique impliquée dans l'activation des acides gras au sein de ces parasites. Chez les eucaryotes, l'activation des acides gras en acyl-coA est une réaction active et obligatoire nécessitant des enzymes clés appelées Acyl-coA synthetases (ACS). Étant donné que les acides gras sont pratiquement impliqués dans tous les aspects de la biochimie cellulaire, leur voie d'activation constituerait donc un talon d'Achille pour la survie du parasite au sein de son hôte. J'ai pu identifié une nouvelle famille de 7 acyl-coA putatives (TgACS) chez Toxoplasma gondii. J'ai pu taggé l'ensemble de ces candidats de manie1re endogène et montre que celles-ci localisent dans des compartiments cellulaires indépendants du parasite. En utilisant des approches de d'inactivation moléculaire et d'analyses lipidomiques basées sur la GC-MS, j'ai réalisé la caractérisation moléculaire de candidate ACS majeurs :

TgACS3. L'inactivation génétique de TgACS3 provoque l'arrêt de la division parasitaire au sein de sa cellule hôte. Le KO de TgACS3 induit une forte réduction de la teneur en phospholipides parasitaires parallèlement à une augmentation significative de la quantité d'acides gras libres, confirmant sa fonction en tant qu'ACS et indiquant son rôle dans l'activation des acides gras nécessaires à la synthèse en masse des phospholipides membranaires du parasite.

Une autre question importante que j'ai abordée lors ma thèse (incluant 2 articles collaboratifs), est l'effet des conditions environnementales / nutritionnelles de l'hôte affectant l'adaptation de la synthèse de novo des acides gras et les capacités de piégeage des FA de l'hôte par le parasite. K'ai mis en place des conditions de culture cellulaire mimant les fluctuations nutritionnelles de l'hôte en faisant fluctuer les quantités de sérume de veau Foetal (FBS), utilisé en tant que source de nutriments dans les mileiux de culture in vitro. En conditions nutritionnelle physiologique, la quantité de nutriments est haute (10%FBS) et permet une croissance soutenue du parasite. Dans le cas de restriction nutritive de l'hôte, les nutriments sont plus faiblement disponibles et impacte directment les taux de division parasitaire au sein de leurs cellules hôtes. Ces mêmes conditions de carence nutritives ont pour conséquence l'augmentation significative de la présence des goutelettes lipidiques au sein du parasite et de sa vacuole parasitophore. Leur présence au sein du parasite est inversement proportionnelle à la quantité du nutriment disponible depuis l'hôte, suggérant une plasticité métabolique du parasite en fonction des conditions nutritives. Lors de l'inactivation de la présence de la protéine TgLIPIN ou de TgACS3, le défaut de croissance qui en résulte fortement aggravé en présence d'un environnement nutritionnel élevé, (10% de FBS). Ceci suggère que la fonction de ces deux protéines est directement liée et impliqué dans l'utilisation des ressources lipidique de l'hôte.

An introduction to the phylum 'APICOMPLEXA'

The phylum apicomplexa resides within the eukaryotic super-group (superphylum) called the 'Alveolata'. Alveolata unites three divergent eukaryotic phyla of unicellular organisms, or protists: Dinoflagellata, Ciliophora (Ciliata), and Apicomplexa (Sporozoa) that have successfully colonized most biotopes on earth. A unifying morphological feature linking the evolution of apicomplexans and dinoflagellates with ciliates, thereby forming the infrakingdom alveolate, is the presence of a unique membrane structure formed of flattened vesicles, called 'cortical alveoli', tightly apposed and maintained underneath the plasma membrane through a network of underlying sub-pellicular microtubules [START_REF] Gould | Alveolins, a new family of cortical proteins that define the protist infrakingdom Alveolata[END_REF]. This triple-membrane structure has evolved to provide a unique platform notably allowing their motility. In apicomplexan parasites the cortical alveoli are dubbed as the inner membrane complex (IMC) located underneath the plasma membrane.

Unlike free-living dinoflagellates and ciliates, apicomplexans have adopted an obligate intracellular parasitic lifestyle. Apicomplexa form five dominant groups of morphologically and ecologically diverse protists including several pathogenic organisms [START_REF] Vot | Handbook of the Protists[END_REF]: a) Gregarinida These include the understudied Gregarina, which are known to inhabit invertebrate (marine, freshwater and terrestrial intestines, coeloms and reproductive vesicles posing as potential threats to insect farms or in laboratory colonies [START_REF] Vot | Handbook of the Protists[END_REF].

b) Piroplasmida

This sub-group derives its name after the pear-shaped intra-erythrocytic stages of the parasites. These include Babesia (B. bovis) and Theileria (T. parva and T. annulata) responsible for cattle-inflicting diseases babesiosis and theileriosis, respectively. These intracellular parasites are transmitted by ticks (Ixodidae) [START_REF] Jalovecka | The complexity of piroplasms life cycles[END_REF].

Piroplasmosis results in low growth, low milk production, death of infected animals and subsequent economic loss (estimated at more than 100 million dollars direct and indirect losses in the USA).

c) Cryptosporidida

Cryptosporidium has been identified as the second most common cause of infant diarrhea in developing countries [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): A prospective, case-control study[END_REF]. This notorious apicomplexan is also responsible for gastrointestinal disease and morbidity within HIV-infected patients. These parasites have a unique epicellular parasitic lifestyle, developing their parasitophorous vacuole (i.e. an intracellular niche which allows its development within the host cell) just on the surface of the invaded host cell.

d) Haemosporidia

Plasmodium, one of the members of Haemosporidia is responsible for enormous human suffering and economic loss through the disease caused, malaria.

e) Coccidia

This group includes parasite Neospora caninum, found worldwide in dogs, cattle, and other mammals, Sarcocytis sp in cattle and Eimeria sp in poultry. One of the most successful coccidian pathogens infecting virtually every mammalian host species, causing

Toxoplasmosis, is Toxoplasma gondii. This parasite can cause serious complications including chorioretinitis (eye damage) and encephalitis in fetuses (congenital toxoplasmosis) and in immunocompromised patients (HIV/AIDS).

This large group of protists encompass up to 6000 described and several other undescribed species.

As described above, this diverse phylum includes unicellular eukaryotes of prime importance in medicine and agriculture. Evolutionary phylogeny roots apicomplexa to photosynthetic flagellates [START_REF] Vot | Handbook of the Protists[END_REF]. This direct link of apicomplexan evolution to the photosynthetic lineage has recently been confirmed by the discovery of photosynthetic protists, Chromerids. Corallicolids, living as mutualists in corals are also the closely related to apicomplexans parasites [START_REF] Janouškovec | A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids[END_REF][START_REF] Kwong | A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes[END_REF].

Some of the striking characteristic features of the Apicomplexa phylum include:

• Complex parasitic lifestyle alternating between sexual and asexual stages in different host types

• Apical complex central to parasite invasion and for the establishment of infection within the host • A non-photosynthetic plastid called 'apicoplast' responsible for essential metabolic functions • A highly reduced genome with a respiring mitochondrion or a non-respiring mitosome (e.g.

Cryptosporidium)

Figure 1 Representation of the evolutionary origin and composition of phylum Apicomplexa. a) A scanning electron microscopic image of Gregarine gamonts. (adapted from [START_REF] Takahashi | Observation by electron microscopy of a gregarine parasite of Antarctic krill: Its histological aspects and ecological explanations[END_REF]. b) Electron microscopic image of Cryptosporidium invading its host (adapted from [START_REF] Elliott | Cryptosporidium Parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface[END_REF]. c) EM image depicting intra-erythrocyte trophozoite stages of Babesia bovis (adapted from [START_REF] Todorovic | Ultrastructure of Babesai bovis (Babes, 1888)[END_REF]. d) Transmission EM image of intracellular Toxoplasma parasite vacuole (adapted from (Amiar et al., 2016). e) TEM of a P. falciparum intra-erythrocytic trophozoite stage (adapted from Francis, Sullivan and Goldberg, 1997).

Apicomplexan parasites with importance in human diseases a) Plasmodium falciparum

This haemosporidian is responsible for malaria, which is one of the most morbidity-causing disease worldwide. According to the WHO malaria report 2019, no significant gain was attained in terms of curbing malaria between the years 2014-2018. Malaria was responsible for 405,000 deaths between 2010-2018, which unfortunately marked children under 5 years of age as the most vulnerable group (approx. 67% malaria deaths) (World Health Organization, 2019). There are at least 5 different species belonging to the genus Plasmodium, including human pathogens P. vivax, P. ovale, P. malariae, P. knowlesi, P. falciparum and rodent malaria pathogens P. berghei, P.

yoelii. Malaria is treatable if diagnosed promptly. The symptoms of malaria can be mild, which include fever, headache, chills, nausea, vomiting and general fatigue. However, in certain cases the manifestations of the disease can be severe including acute kidney failure, enlargement of spleen, metabolic acidosis, hypoglycemia and severe anemia. Cases where the parasite crosses the blood brain barrier results in cerebral malaria and ultimately death without any known treatment.

Existing effective treatment against P. falciparum caused malaria is artemisinin combination therapy (ACT), which includes artemisinin-based compounds in combination with companion drugs like lumefantrine, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, piperaquine and chlorproguanil/dapsone. However, its less notorious siblings like P. vivax and P. ovale are still treated using only chloroquine. The emerging resistance against artemisinin suggests the pressing need of novel drugs [START_REF] Dondorp | Artemisinin Resistance in[END_REF].

b) Toxoplasma gondii

T. gondii, responsible for Toxoplasmosis, inhabits approximately one third of the human population worldwide. The seropositivity of this polyxenous parasite amongst humans ranges from 15-70% [START_REF] Tenter | Toxoplasma gondii: From animals to humans[END_REF]. Till date, the drugs available for Toxoplasma treatment include sulfadiazine and pyrimethamine, mainly targeting parasite's folic acid metabolism. These drugs are active only against the symptomatic acute stage of the parasite known as tachyzoite. The chronic stage bradyzoites of Toxoplasma can persist within their hosts for lifetime, as they remain asymptomatic. Immunocompromised individuals like patients with HIV/AIDS or autoimmune disorders and fetuses can face severe consequences of Toxoplasmosis which results in damage to the brain, eyes, or other organs. Despite being well studied, there are still no vaccines effective against this disease.

Life cycle of apicomplexan parasites

The life cycle of apicomplexans is rather complex and occurs within two different hosts types.

This thesis is focused on two apicomplexan parasites responsible for inflicting morbidity in humans, Toxoplasma gondii and Plasmodium falciparum. The life cycle of coccidians and haemosporidians comprises both asexual division (merogony) and sexual division (gamogony) [START_REF] Vot | Handbook of the Protists[END_REF].

Life cycle of Plasmodium falciparum

This notorious pathogen has a complex life cycle balancing between female Anopheles mosquito and a vertebrate host. The life cycle begins with a blood feed by an infected female Anopheles mosquito where it injects sporozoites life stages into the dermis of the vertebrate host. Some of these sporozoites that escape the host immune response within the dermis are successfully able to invade liver cells (i.e. hepatocytes) via bloodstream. This invasion initiates the liver stage of Plasmodium life cycle, which is an asymptomatic step, allowing the exponential multiplication of the parasite population. The active invasion of a single sporozoite into a hepatocyte is preceded by the traversal of these sporozoites through various hepatocytes involving formation of a transient vacuole.

Once the sporozoite has invaded a hepatocyte, it converts to an exo-erythrocytic form (EEF) over the subsequent 2-10 days after invasion. These EEFs undergo massive asexual replication during which the karyokinesis precedes cytokinesis, called 'schizogony' culminating in the release of up to 40,000 merozoites per hepatocyte into the bloodstream by budding and active egress of parasite-filled vesicles called merosomes [START_REF] Sturm | Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids[END_REF]. The release of merozoites initiates the symptomatic blood stage of the parasite life cycle. These merozoites next encounter and infect red blood cells (RBCs). The RBC invasion by the merozoites is a dynamic multistep process (attachment, apical reorientation and actual invasion) that is complete within 2 min [START_REF] Cowman | Malaria: Biology and Disease[END_REF]. Once erythrocyte infection is established, parasite undergoes another round of schizogony. The invaded parasite then undergoes morphological, cellular and metabolic changes to accomplish the asexual blood stage division cycle, through parasite maturation into stages called ring, trophozoite and schizont over the following 48 h. Such division cycle allows the generation of 16-32 merozoites per erythrocyte. The end of this process is marked by the destructive release of these merozoites into the blood stream initiated by the active egress of parasites out of the RBC to access new host cells. During the several rounds of schizogony in the bloodstream, few parasites commit to sexual differentiation and develop into gametocytes. After commitment, it takes up to 11 days for the gametocytes to mature into infectious forms ready to be transmitted to mosquitos. Upon ingestion through the Anopheles blood feed, these gametocytes develop into female non-motile macrogamete and male exflagellated motile microgametes, within the mosquito mid-gut, likely upon the sensing of environmental signals (like temperature).

Furthermore, mating occurs by fusion of micro-and macrogamete resulting in the formation of a zygote, which transforms over next 24 hr into a motile ookinete. The ookinete traverses the mosquito midgut epithelium and encysts to become an oocyst where asexual sporogenic replication occurs [START_REF] Kuehn | The coming-out of malaria gametocytes[END_REF]. Following oocyst rupture, thousands of motile sporozoites are released into the hemocoel which then pass into salivary glands from where they can be injected into the next human host and restart the deadly life cycle. Illustration of complete life cycle is described in figure.2. [START_REF] Cowman | Malaria: Biology and Disease[END_REF]) and sexual development involving life stages female/male gametes, ookinetes, oocysts and sporozoites within female Anopheles mosquito (b), adapted from (Aly, Vaughan, and Kappe 2009). The initiation of asexual life cycle within human host is initiated by the bite of Anopheles injecting sporozoites into the dermis. This is followed by their transport to liver known as the pre-erythrocytic stage where they divide by schizogony (over a period of 10 days) to generate tens of thousands of daughter merozoites are released in packets of merosomes into the bloodstream. Within the bloodstream, these merozoites invade red blood cells carrying out their erythrocytic life stage, a cycle of which lasts for over 48 h. Next, a 15 days period leading to intra-erythrocytic gametocyte development follows which enters the peripheral circulation for ingestion by a mosquito for further transmission. The first part of sexual life cycle occurs in the mosquito midgut where the gametocytes emerge as male and female gametes followed by their fusion into a zygote which transforms into ookinete over a period of 24 h. the ookinete encysts to become oocyst where following sporogony over a period of 10-14 days, thousands of sporozoites are produced. These sporozoites traverse to enter into mosquito salivary gland from where they are transmitted to mammalian host over next blood meal.

Life cycle of Toxoplasma gondii

Toxoplasma gondii was first described in 1908 by Nicolle and Manceaux while working in North Africa, on a semidesert rodent, the common gundi (Ctenodactylus gundi) [START_REF] Black | Lytic Cycle of Toxoplasma gondii[END_REF]. This intracellular parasite replicates within a nucleated host cell with the goal of producing infectious progeny for further survival and dissemination. The complex life cycle of this apicomplexan parasite is split between two host types: an intermediate and a definitive host (illustration: figure . 3).

1) Intermediate host: Toxoplasma asexual life stage

The intermediate host covers a wide range of warm-blooded animals, including humans, where

Toxoplasma undergoes asexual replication and further differentiation into transmissible forms.

Within the intermediate host, the parasite is able to fancy two different life forms: fast replicating 'tachyzoites' and slow dividing 'bradyzoites'. The tachyzoite is virtually able to infect any nucleated cell at the advent of T. gondii lytic cycle. Although, current data suggest that on top of this large repertoire of host cell type, the parasite can choose and/or use the natural capacities of the host cell to its advantage to improve its dissemination/propagation/dormancy. This asexual life cycle is often called the lytic cycle which proceeds via five cellular steps: attachment, active invasion, vacuole formation notably through the discharge of secretory organelles, replication involving all complex metabolic interactions with the host cell, and active egress [START_REF] Black | Lytic Cycle of Toxoplasma gondii[END_REF]. After invading the host cell, a tachyzoite surrounds itself with a parasitophorous vacuole (PV) that is the niche and site for interaction between the parasite and its host cell.

Replicative tachyzoites undergo several rounds of division, with a generation time of approx. [6][7][8] h before the final egress out of their inhabitant host cell to further invade its surrounding host cells.

The terminology and cellular process used for this replicative process, where daughter progeny is formed within the boundaries of the mother cell that gets consumed at the end of division, is called 'endodyogeny' (Francia and Striepen, 2014;[START_REF] White | Apicomplexa Cell Cycles: Something Old, Borrowed, Lost, and New[END_REF]. Under immune pressure from the host, the acute phase tachyzoites can commit, convert and differentiate into slow growing bradyzoites, finally forming a structure called tissue cyst, which constitutes a resistant and dormant niche filled with bradyzoites. Normally, the tissue cysts appear 7-10 days post infection, which defines the so-called chronic life stage of this parasite. The dormant cyst forms usually occur within the central nervous system and muscle tissue, where they can survive within host for lifetime. Congenital infection occurs in case of vertical transmission of these tachyzoites from maternal blood into fetal tissues [START_REF] Dubey | The History and Life Cycle of Toxoplasma gondii. Second Edi, Toxoplasma Gondii: The Model Apicomplexan -Perspectives and Methods: Second Edition[END_REF].

2) Definitive host: Toxoplasma Sexual life stage

In T. gondii life cycle, the sexual stage is restricted to a feline host, that can acquire Toxoplasma through ingestion of contaminated food. The ingested Toxoplasma invades the feline intestinal epithelium where it differentiates into five morphologically distinct forms of schizont [START_REF] Pittman | Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections[END_REF]. These schizonts then differentiate into merozoites, which is regarded as the first sexual stage of the parasite life cycle. Merozoites then undergo 2-4 rounds of division to further differentiate into microgametes and macrogametes. This process of division where the nucleus undergoes multiple rounds of division prior to cytokinesis is called 'schizogony' (Ferguson et al., 1974;Francia and Striepen, 2014). These gametes fuse together to form diploid oocyst. These oocysts are shed via cat's feces into the environment, where they can survive moderate conditions due to their possession of a thick impermeable wall. Cats excrete up to 20 million oocysts per day, after approx. 3-10 days of acquiring Toxoplasma infection. Under favorable environmental conditions, these oocysts undergo mitotic and meiotic divisions to produce haploid sporozoites encysted by the sturdy oocyst wall.

Toxoplasma transmission between various hosts occurs via: a) Faeco-oral route, by ingestion of food contaminated with Toxoplasma oocysts b) Congenital acquisition, vertical transmission of parasites from infected mother to fetus Since, the results of my thesis are majorly focused on T. gondii, therefore further description of parasite morphology and establishment of infection within the host is focused on Toxoplasma.

Establishment of an infectious niche: active host cell invasion by T. gondii

Host cell invasion by Toxoplasma is an active process, unlike several other eukaryotic pathogens that embark upon phagocytic engulfment by the host. These parasites employ their actin-myosin machinery for aiding the active entry into their hosts [START_REF] Carruthers | Host cell invasion by the opportunistic pathogen Toxoplasma gondii[END_REF].

Toxoplasma invasion begins with a reversible attachment of the parasite to the host cell surface, a process called 'gliding motility'. Within Toxoplasma, apically located secretory organelles, micronemes and rhoptries act as the main drivers in the process host cell invasion. For instance, the initial binding of the parasite to host cell membrane results in apical release of the micronemal protein MIC2 [START_REF] Carruthers | Sequential protein secretion front three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts[END_REF]. The second step is initiated by the discharge of rhoptry proteins in order to form a nascent parasitophorous vacuole. The rhoptries empty their contents apically during the process of host invasion i.e., beginning with the RONs and thereafter

ROPs. During the process of invasion, a constriction or tight association, called the moving junction (MJ), is formed between the parasite and host cell membrane [START_REF] Mordue | Proteins on the Basis of Their Membrane Anchoring[END_REF][START_REF] Alexander | Identification of the moving junction complex of Toxoplasma gondii: A collaboration between distinct secretory organelles[END_REF]. This MJ associated constriction around an invading parasite migrates from the anterior towards the posterior end. The MJ also serves to exclude all the host cell proteins and parasite secreted micronemes from entering the parasite vacuolar space within its host. The moving junction is formed with a complex of the micronemal protein AMA1 and RONs (RON2, 4,5 and 8) within the plasma membrane of the parasite [START_REF] Besteiro | Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion[END_REF]. At the end of the invasion process when the PV formation is complete, the dense granules release their contents called GRA proteins into the PV and beyond to the host cell nucleus [START_REF] Hakimi | Toxoplasma's ways of manipulating the host transcriptome via secreted effectors[END_REF][START_REF] Mercier | Toxoplasma secretory granules: One population or more?[END_REF]. Dense granule proteins (GRA) play important roles in the interaction of the parasite with its host via PVM as well as survival within the host through rewiring of the host cell gene expression.

Ultrastructural morphology of Toxoplasma tachyzoite

Toxoplasma derives its name based on its shape, from a Greek work toxon, meaning "bow". A typical Toxoplasma tachyzoite is crescent-shaped ranging in the size of 2-6 µm [START_REF] Dubey | Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts[END_REF]).

The apical complex: machinery guiding the invasion process in T. gondii

The phylum apicomplexa withholds its name due to the presence of an 'apical complex' which is central to parasite invasion and subsequent proliferation within its host [START_REF] Hu | Cytoskeletal components of an invasion machine -The apical complex of Toxoplasma gondii[END_REF]. The apical complex is a remarkable structure comprising of a) spirally arranged assembly of fibers called conoid, b)polar ring, located at the parasite apex, which serves as the site for origination of subpellicular microtubules, c) two intraconoidal microtubules and d) the preconoidal rings.

Toxoplasma has several apically located regulated secretory organelles which include micronemes and rhoptries. Micronemes are small rod-shaped electron dense structures. The micronemes released their contents early on during the process of attachment-invasion, following which there is secretion of rhoptries as the invasion process proceeds. The rhoptries appear as club shaped structures in electron micrographs of T. gondii. This parasite typically has up to 8-12 rhoptries in total which are composed of a bulbous body structure which tapers into a thin duct-like rhoptry neck extending towards the conoid. The rhoptry neck proteins are referred to as RONs whereas the ones residing in bulbous body area are called ROPs. Rhoptries are known to be rich in proteins as well as lipids which they release alongside their secretion during the time of invasion. There are other cytoplasmic secretory organelles called dense granules that are released towards the end after completion of the process of invasion [START_REF] Black | Lytic Cycle of Toxoplasma gondii[END_REF].

2) Toxoplasma pellicle: the outer membranes surrounding the parasite

The parasite pellicle is a trilaminar structure comprising of the outer plasma membrane and an inner membrane complex (IMC) comprising two continuous membranes [START_REF] Dubey | Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts[END_REF]. IMC is a unique cytoskeletal organelle which comprises of two distinct elements: a sac of flattened vesicles beneath the parasite plasma membrane called alveoli and a supporting rigid network of intermediate filaments [START_REF] Mann | Characterization of the subpellicular network, a filamentous.pdf[END_REF][START_REF] Chen | Novel components of the toxoplasma inner membrane complex revealed by BioID[END_REF]. The IMC imbibes a highly dynamic organization crucial for parasite development. The IMC of the mother parasite acts as the scaffold used by daughter cells for undergoing the process of endodyogeny (Ouologuem and Roos, 2014). The daughter IMC elongation is based on the recycling of maternal IMC membranes, after emergence of daughters from the mother cell.

Figure 6

The basic pellicle organization Toxoplasma tachyzoite is characterized by alveolar sacs (ALV) associated with the inner membrane complex beneath the plasma membrane (in yellow) supported by a membrane skeleton (MS) and associated with microtubules structures (red) (adapted from [START_REF] Gould | Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates[END_REF] 3) Metabolically active organelles: Apicoplast and Mitochondria Similar to their algal ancestors these parasites also possess a single apicoplast and mitochondrion.

Apicoplast:

It is a relict chloroplast present in several apicomplexan parasites, acquired in an event involving secondary endosymbiosis. The primary endosymbiotic event that gave rise to chloroplast in plants and algae arose from engulfment of Cyanobacterium by a eukaryotic heterotroph. This was followed by a second endosymbiotic event wherein plastids of chromalveolates were derived from red algae [START_REF] Lim | The evolution, metabolism and functions of the apicoplast[END_REF][START_REF] Van Dooren | The Algal Past and Parasite Present of the Apicoplast[END_REF]. Based on its evolutionary origin apicoplast is surrounded by four membranes enriched with phospholipids instead of galactolipids as their algal ancestors [START_REF] Botté | Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites[END_REF]. Apicoplast has time n again proven itself to be parasites Achilles heel due to its role as parasite's central metabolic hub. This special organelle harbors several metabolic processes crucial for the intracellular development of apicomplexan parasites (van [START_REF] Van Dooren | The Algal Past and Parasite Present of the Apicoplast[END_REF].

i)

Type-II Fatty acid biosynthesis/ FASII pathway: Gene knockout studies of several important enzymes involved in FASII pathway have proven the indispensability of this metabolic pathway for T. gondii. The apicoplast FASII contributes to the total cellular pool of short chain fatty acids (myristate, palmitate) which are further used for membrane biogenesis aiding parasite cell division and hence survival within their host (Mazumdar et al., 2006;Ramakrishnan et al., 2012;Amiar et al., 2016).

ii) Isoprenoid precursor assembly pathway: The central precursor required for isoprenoid biosynthesis is isopentenyl pyrophosphate (IPP). Unlike mammalian cells, the IPP within apicomplexans is synthesized via a non-mevalonate pathway, involving methylerythritol phosphate (MEP), localized within parasite apicoplast.

Genetic ablation of the last step of the MEP pathway involving enzyme LytB is lethal to the Toxoplasma tachyzoites [START_REF] Nair | Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii[END_REF]. Isoprenoids act as components of membrane lipids and mitochondrial electron chain coenzyme Q.

These compounds also participate in post-translational modifications of several proteins imparting important functions like protein-protein interactions (van [START_REF] Van Dooren | The Algal Past and Parasite Present of the Apicoplast[END_REF].

iii) Iron-Sulphur cluster biosynthesis: Iron-Sulphur (Fe-S) clusters act as prosthetic groups for proteins involved in various redox reactions (van [START_REF] Van Dooren | The Algal Past and Parasite Present of the Apicoplast[END_REF]. The apicoplast based Fe-S proteins are involved in IPP and fatty acid biosynthetic pathway, thereby suggesting the importance of this metabolic pathway in T. gondii.

iv)

Heme biosynthesis pathway: Heme is an essential prosthetic group that functions in many cellular redox reactions including the mitochondrial electron transport chain ( [START_REF] Van Dooren | The use and abuse of heme in apicomplexan parasites[END_REF]. Apicomplexan parasites harbor an unusual pathway of heme biosynthesis spanning three different cellular compartments-cytosol, apicoplast and mitochondria [START_REF] Kořený | Make It, Take It, or Leave It: Heme Metabolism of Parasites[END_REF][START_REF] Bergmann | Toxoplasma gondii[END_REF]. Recent study provides the first evidence for presence of a functional heme biosynthesis pathway within Toxoplasma apicoplast [START_REF] Bergmann | Toxoplasma gondii[END_REF].

Mitochondria:

This organelle acts as reserve of essential central carbon metabolic pathways like the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC). Chemical inhibition of mitochondrial TCA cycle with sodium fluoroacetate (NaFAc) is lethal to Toxoplasma tachyzoites [START_REF] Macrae | Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of toxoplasma gondii[END_REF]. T. gondii is able to utilize both glucose and glutamine as substrates via the TCA cycle.

The catalytic conversion of glycolytic intermediate pyruvate into acetyl coA for further utilization in mitochondrial TCA cycle is facilitated by a branched chain ketoacid dehydrogenase (BCKDH) complex. BCKDH is a functional replacement of mitochondrial PDH in apicomplexan parasites and essential for their intracellular growth [START_REF] Oppenheim | BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei[END_REF]. Another enzyme succinyl coA synthetase, catalyzing the seventh step of TCA cycle succinyl-CoA to succinate, was found to be dispensable for parasite growth (Fleige et al., 2007). A probable reason for this unexpected dispensability could be the leaky expression of the enzyme under control of the anhydrotetracycline regulatable element.

A potential metabolic link between Toxoplasma apicoplast and mitochondria was established by

showing that the use of a specific inhibitor of TCA cycle, NaFAc resulted in partial reduction in apicoplast FASII biosynthesis. The reason for the same was justified because of a shunt in which mitochondrial citrate is transported to the apicoplast and converted to a-ketoglutarate with regeneration of NADPH (MacRae et al., 2012) 4)Other organelles covering the endomembrane system include the posteriorly located nucleus, the tubular network formed by the endoplasmic reticulum extending from the nuclear envelope and the Golgi complex [START_REF] Nishi | Organellar dynamics during the cell cycle of Toxoplasma gondii[END_REF]. The ER is predominantly located basal end of the parasite while as the Golgi is adjacent to the apical end of the parasite nucleus [START_REF] Hager | The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii[END_REF]. ; Rhoptry neck protein ROP1 and dense granule protein GRA7 was ( adapted from [START_REF] Wang | Three toxoplasma gondii dense granule proteins are required for induction of lewis rat macrophage pyroptosis[END_REF]; conoid (adapted from (Katris et al., 2014) ; Golgi marker GRASP (adapted from [START_REF] Pfluger | Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii[END_REF]; mitochondrial marker TOM40 (adapted from ( [START_REF] Van Dooren | The import of proteins into the mitochondrion of toxoplasma gondii[END_REF] ; microneme marker MIC5 (adapted from Huynh, Boulanger and Carruthers, 2014)

LIPID METABOLISM IN APICOMPLEXA: Biosynthesis, Uptake and Recycling

The survival of these parasites within host is predominantly dependent on lipids which play an essential role by regulating metabolic flux, acting as signaling molecules, storage fuels and structural building blocks of membranes. These parasites meet their high demand of lipids through de novo synthesis within apicoplast as well as via copious salvage directly from the host and extracellular environment (Coppens, 2006(Coppens, , 2013)). This raises the prospect that lipid homeostatic, trafficking and remodeling pathways in the parasite may abound as potential drug targets. For instance, apicoplast resident fatty acid biosynthetic enzymes have been successfully exploited as antimicrobial targets against Toxoplasma infections. This section summarizes different lipid species present in two important apicomplexan parasites T. gondii and P. falciparum:

Phosphatidylcholine

Phosphatidylcholine (PC) is a neutral glycerophospholipid accounting for the major component of total complex lipids. Quantification of total phospholipid profile within apicomplexa reveals high abundance of phosphatidylcholine in T. gondii (40%-70%) as well as in P. falciparum (40%-50%) [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF]Welti et al., 2007;[START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF].

Toxoplasma tachyzoites can synthesize PC via de novo Kennedy pathway involving a cytoplasmic choline kinase which utilises choline up taken from external environment [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF][START_REF] Sampels | Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii[END_REF]. Selective disruption of PC biosynthesis by supplementation of a choline analog, dimethylethanolamine was able to cause dramatic parasite growth arrest.

Dimethylethanolamine interferes with choline uptake and subsequent metabolism to phosphatidylcholine, resulting in inhibition of parasite intracellular replication due to inhibition of PC biosynthesis or massive accumulation of toxic intermediate phosphatidyldimethylethanolamine [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF].

Despite the presence of CDP-choline/Kennedy pathway for major PC biosynthesis, P. falciparum also employs an alternative pathway called serine-decarboxylase-phosphoethanolaminemethyltransferase (SDPM) pathway which uses host serine and ethanolamine as precursors (Elabbadi, Ancelin and Vial, 1997;[START_REF] Pessi | Pathways for phosphatidylcholine biosynthesis: targets and strategies for antimalarial drugs[END_REF][START_REF] Witola | Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects[END_REF]. This alternative pathway begins with the decarboxylation of host-derived serine to ethanolamine via serine decarboxylase (PfSD). The next step in catalyzed by a phosphoethanolamine methyltransferase (PfPMT) in a three-step methylation reaction starting from substrate phosphoethanolamine to phosphocholine which is further incorporated into phosphatidylcholine (PC) [START_REF] Pessi | A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation[END_REF][START_REF] Pessi | Pathways for phosphatidylcholine biosynthesis: targets and strategies for antimalarial drugs[END_REF]. The loss of PfPMT by gene knockout studies results in strong growth defects alongside abrogation of PC biosynthesis via SDPM pathway within P. falciparum [START_REF] Witola | Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects[END_REF]. However, unlike P. falciparum, Toxoplasma cannot use ethanolamine as a substrate for PC biosynthesis due to the absence of the required methyltransferase enzyme activity [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF].

Phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a neutral, second most abundant phospholipid in eukaryotic cells, normally present in the inner bilayer of the plasma membrane. Expectedly, PE is also the second most abundant phospholipid in T. gondii (10%-20%) and P. falciparum (15%-25%) (Welti et al., 2007;[START_REF] Hartmann | Phosphatidylethanolamine synthesis in the parasite mitochondrion is required for efficient growth but dispensable for survival of toxoplasma gondii[END_REF][START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF].

In T. gondii, PE biosynthesis occurs differentially at several organellar sites, in the mitochondrion and in the PV by decarboxylation of phosphatidylserine, and in the ER by fusion of CDPethanolamine and diacylglycerol [START_REF] Gupta | The obligate intracellular parasite Toxoplasma gondii secretes a soluble phosphatidylserine decarboxylase[END_REF][START_REF] Hartmann | Phosphatidylethanolamine synthesis in the parasite mitochondrion is required for efficient growth but dispensable for survival of toxoplasma gondii[END_REF]. Mitochondrial PE biosynthesis is catalyzed by the enzyme phosphatidylserine decarboxylase (TgPSD1mt). The depletion TgPSD1mt impacts parasite's optimal growth, which is rescued by the addition of exogenous ethanolamine. However, despite the simultaneous knockdown of TgPSD1 and ethanolamine-depletion the parasites can still egress and form plaques, albeit reduced in terms of area in comparison to wild type parasites [START_REF] Hartmann | Phosphatidylethanolamine synthesis in the parasite mitochondrion is required for efficient growth but dispensable for survival of toxoplasma gondii[END_REF]. This suggest that the parasite has employed other routes for PE biosynthesis including the high possibility of direct scavenging from its metabolically enriched host. Toxoplasma also secretes a phosphatidylserine decarboxylase (TgPSD1) localizing to the dense granules, into the parasite vacoular space [START_REF] Gupta | The obligate intracellular parasite Toxoplasma gondii secretes a soluble phosphatidylserine decarboxylase[END_REF].

Based on biochemical activity confirmation of TgPSD1, it can be speculated that this enzyme contributes to PE biosynthesis within PV, possibly utilizing host derived precursors.

The PE biosynthesis in P. falciparum is also branched into CDP-ethanolamine/Kennedy pathway and the serine decarboxylation pathway (as described above) (Elabbadi, Ancelin and Vial, 1997;[START_REF] Kilian | Role of phospholipid synthesis in the development and differentiation of malaria parasites in the blood[END_REF].

Phosphatidylserine

Phosphatidylserine (PS) despite being less than 10% in the total abundance of phospholipids, is still an important anionic lipid. Due to its negative charge, PS is able to impart unique biophysical properties to the membranes and also participates in important biological processes like apoptosis in various eukaryotic cells. In terms of abundance, PS constitutes 5%-10% of the total phospholipid species in both T. gondii and P. falciparum (Welti et al., 2007;[START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF].

Intriguingly, phosphatidylserine in T. gondii has been linked to a mechanism of host immune evasion called apoptotic mimicry [START_REF] Dos Santos | Phosphatidylserine exposure by Toxoplasma gondii is fundamental to balance the immune response granting survival of the parasite and of the host[END_REF]. Briefly, during this process Toxoplasma expresses PS on the surface thereby decoying itself as an apoptotic cell inside its host macrophages.

As a result, the parasite infected macrophages inhibit the synthesis of nitric oxide (NO) via NO synthase degradation, thereby allowing parasite persistence within. Interestingly, Toxoplasma utilizes serine as a substrate for PS biosynthesis through two different enzymes functionally behaving as phosphatidylserine synthases, TgPSS (phosphatidylserine synthase) and TgPTS (phosphatidylthreonine synthase) [START_REF] Arroyo-Olarte | Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence[END_REF]. TgPSS, with strict serine-substrate specificity, localizes towards the parasite ER/mitochondrion intersection.

In P. falciparum PS acts as an important pathogenicity factor by facilitating erythrocyte cytoadherence (Eda and Sherman, 2002). Micro vesicles derived from the blood sample of P.

falciparum infected patients been have shown to have elevated levels of phosphatidylserine [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. The malarial parasite synthesizes PS using host derived serine, most of which is obtained from parasite mediated host erythrocyte hemoglobin degradation (Wein et al., 2018). As described above PS also serves as a precursor of PE via serine decarboxylation pathway in both T.

gondii and P. falciparum (Elabbadi, Ancelin and Vial, 1997;[START_REF] Hartmann | Phosphatidylethanolamine synthesis in the parasite mitochondrion is required for efficient growth but dispensable for survival of toxoplasma gondii[END_REF][START_REF] Kilian | Role of phospholipid synthesis in the development and differentiation of malaria parasites in the blood[END_REF].

Phosphatidylthreonine

Phosphatidylthreonine (PTh) is usually a minor component of the total phospholipid constituent in various eukaryotic cells. Recent study described the existence of Phosphatidylthreonine as a major phospholipid (3-4 times more than phosphatidylserine) in T. gondii facilitating a 'lipidmediated virulence' within its host (Arroyo-Olarte and Gupta, 2016). The parasite employs a novel enzyme phosphatidylthreonine synthase (TgPTS) for generation of PTh within the endoplasmic reticulum. The TgPTS has evolutionarily evolved from TgPSS as both the enzymes are capable for PS synthesis in the presence of serine as a substrate. Unlike, TgPSS, the presence of threonine induces phosphatidylthreonine biosynthesis by the enzymatic action of TgPTS. Genetic disruption of TgPTS (Δtgpts) impairs the parasite lytic cycle and attenuates its growth within murine model.

Lipidomic analysis of parasites lacking

TgPTS indicate a significant decline in the PTh levels with a concomitant accumulation of phosphatidylserine. The rescue of strong growth defect and PTh synthesis is attained by complementation of Δtgpts parasites with exogenous copy of TgPTS-HA.

The main fatty acid components of the T. gondii PTh species included C20:1 and C20:4 (Arroyo- et al., 2015). The authors of this study further speculate the possible involvement of PTh influencing the calcium flux across the plasma membrane and/or calcium storage organelles (Arroyo-Olarte and Gupta, 2016).

Olarte

Phosphatidylinositol and related phosphoinositides

Phosphatidylinositol (PI) is a negatively charged important constituent of membrane phospholipids. Phosphate derivatives of phosphatidylinositol, called phosphoinositides have more critical functions within a cell, such as signaling, cell-cell communication, intracellular trafficking.

PI comprises approx. 5% of the total phospholipid abundance in both T. gondii and P. falciparum (Welti et al., 2007;[START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. In Toxoplasma, phosphatidylinositol-3-monophosphate (PI3P) has been shown to be associated with apicoplast protein-shuttling vesicles. Chemical inhibition of PI3P synthesizing kinase, resulted in aberrant apicoplast morphological development suggesting the role of this phospholipid for apicoplast biogenesis [START_REF] Tawk | Phosphatidylinositol 3-monophosphate is involved in Toxoplasma apicoplast biogenesis[END_REF]. A Toxoplasma phosphatidylinositide-phospholipase C (TgPI-PLC), catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to D-myo-inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) at the plasma membrane of the parasite (Fang, Marchesini and Moreno, 2006). Recombinant protein of TgPI-PLC showed a preference for phosphatidylinositol (PI) as a substrate rather than PIP2. Another study showed that this protein was localizing to both plasma membrane and cytoplasm within the parasite, suggesting its multiple roles in lipid regulation (Bullen et al., 2016).

In P. falciparum, an enzyme generating 3' phosphorylated phosphoinositides, phosphoinositide-3-kinase (PI3K) is actively transported across the PVM into its erythrocyte host. Using PI3K inhibitors wortmannin and LY294002, the enzyme and its product were shown to be functionally involved in endocytic trafficking of hemoglobin from the host to the food vacuole within the parasite. This enzymatic function is necessary for the parasite pathogenesis as it allows degradation of the toxic host hemoglobin within the parasite food vacuole to provide amino acid sources for its growth [START_REF] Vaid | PfPI3K, a phosphatidylinositol-3 kinase from Plasmodium falciparum, is exported to the host erythrocyte and is involved in hemoglobin trafficking[END_REF]. MMV390048, a drug effective against all life stages of malarial parasites specifically targets a phosphatidylinositol 4-kinase within the parasite [START_REF] Paquet | Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4kinase[END_REF]. The sugar-derivates of phosphatidylinositol like glycosylphosphatidylinositol (GPI) have been identified as dominant toxins existing as free or antigen linked molecules on the surface of malaria parasite [START_REF] Debierre-Grockiego | Fatty acids from Plasmodium falciparum down-regulate the toxic activity of malaria glycosylphosphatidylinositols[END_REF]. Plasmodium falciparum uses de novo synthesized as well as host-scavenged myo-inositol for the synthesis of bulk phosphatidylinositol [START_REF] Macrae | Plasmodium falciparum is dependent on de novo myo-inositol biosynthesis for assembly of GPI glycolipids and infectivity[END_REF]. The scavenged myo-inositol is however directly channeled towards biosynthesis of glycophosphatidylinositol (GPI).

Cardiolipin

Cardiolipin (CL) is basically a diphosphatidyl glcyerolipid bearing four fatty acyl chains and hence double negative charge. The occurrence of cardiolipin is mostly in prokaryotic plasma membrane and eukaryotic mitochondrial membranes [START_REF] Schlame | Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes[END_REF]. The synthesis of CL within eukaryotes begins with conversion of phosphatidic acid (PA) to cytidine diphosphate-diacylglycerol (CDP-DAG). The final reaction involves an enzyme cardiolipin synthase which catalyzes the transfer of phosphatidyl group from CDP-DAG to another lipid molecule phosphatidylglycerol (PG) resulting in the formation of cardiolipin [START_REF] Schlame | Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes[END_REF][START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF].

The presence of a putative cardiolipin synthase candidate gene (TGGT1_309940) is indicative of functional de novo cardiolipin biosynthesis pathway in Toxoplasma [START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF]. A recent study showed that a fatty acid transporter, acyl-coA binding protein-2 (TgACBP2) actively participates in cardiolipin metabolism and subsequently maintains mitochondrial homeostasis within type-II strain of T. gondii. Metabolic analysis of TgACBP2 depleted type II prugnaid strain using LC-MS showed significant reduction in cardiolipin abundance, esp. with fatty acyl moieties C72:7, C74:9, C74:8, C74:7, C76:9, C78:11, C78:9, C78:8, C82:11, and C82:0. Interestingly, there are reports of increased prevalence of anti-cardiolipin antibodies in patients inflicted with malaria in Asia and Africa [START_REF] Consigny | High prevalence of co-factor independent anticardiolipin antibodies in malaria exposed individuals[END_REF].

Phosphatidylglycerol

Phosphatidylglycerol (PG) is an anionic phospholipid less abundant in eukaryotes but highly abundant in bacterial membranes. PG in P. falciparum makes up almost 2%-3% of the total phospholipids [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. The levels of PG increase in P. falciparum infected erythrocytes from undetectable levels to approx. 4% [START_REF] Tran | Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum[END_REF]. The same phospholipid is reduced from 4% to 1% upon maturation of asexual stages to sexual stage gametocytes. There is not much literature available for the functions and biosynthesis of PG in T. gondii, however it acts an important precursor for the biosynthesis of another important mitochondrial lipid cardiolipin.

Cytidine diphosphate-diacylglycerol

Cytidine diphosphate-diacylglycerol (CDP-DAG) is a nucleotide of prime importance in lipid metabolism. This high energy intermediary liponucleotide that serves as a precursor for several other phospholipids, however, remains undetectable in lipid composition of various cells. CDP-DAG in Toxoplasma is synthesized in two ways, in the ER by a eukaryotic-type CDP-DAG synthase1 (TgCDS1) and in the apicoplast by a prokaryotic-type CDP-DAG synthase2 (TgCDS2) (Kong et al., 2017). Genetic knockdown of TgCDS1 was lethal to Toxoplasma tachyzoites and resulted in a significant reduction in the levels of phosphatidylinositol and phosphatidylserine. On contrary, complete knockout of TgCDS2 resulted in selective impairment of phosphatidylglycerol biosynthesis. Overall, a deficit CDP-DAG synthesis pathway within T. gondii results in attenuated parasite virulence within mice.

P. falciparum has been reported to have a CDP-DAG synthase with split protein properties [START_REF] Shastri | Plasmodium CDP-DAG synthase: An atypical gene with an essential N-terminal extension[END_REF]. The same study demonstrated that a knockout of N-terminal extension of PfCDS was lethal to parasite blood stages, suggesting the importance of this liponucleotide intermediate in the phospholipid metabolism of malaria parasites.

Sphingomyelin

Sphingolipids usually comprise of a long chain or spinghoid bases (e.g. sphingosine or ceramide)

which is attached to fatty acyl moiety via amide bond.

In comparison to their host mammalian cells, T. gondii has an overall lesser abundance (approx.

1%) of sphingolipids (Welti et al., 2007). Intriguingly, this parasite has a relatively high abundance of another unusual sphingolipid called ceramide phosphoethanolamine (EPC). EPC comprises approx. 2% of the total polar lipid content in T. gondii. Evidence of de novo sphingolipid biosynthesis in T. gondii was first demonstrated by the bioinformatic identification of a sphingolipid synthase (TgSLS). The enzyme TgSLS was also biochemically validated by its ability to functionally complement a yeast mutant lacking inositol phosphorylceramide (IPC) synthase [START_REF] Pratt | Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii[END_REF]. Metabolic labelling studies also provided experimental evidence of the uptake of tritiated serine and galactose by T. gondii for de novo glycosphingolipid biosynthesis [START_REF] Azzouz | Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii[END_REF]. Despite the presence of a de novo biosynthetic pathway this parasite also relies on host scavenged sphingolipids. Toxoplasma accrues sphingolipids from the host in a Golgi dependent manner, with the help of Rab-coated vesicles. Experimental evidence of the same was provided by colocalization of vesicles, marked with Rab14, Rab30, or Rab43, with host derived sphingolipids in the vacuolar space surrounding the parasites [START_REF] Romano | Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole[END_REF]. These parasites replicate slowly in the host cells that are impaired in sphingolipid biosynthesis. For instance, use of myriocin, an inhibitor of serine palmitoyl transferase, in infected host cells was able to decrease the rate of parasite replication in a dose dependent manner, suggesting the importance of sphingolipid uptake in T. gondii.

Sphingolipids encompass the second most abundant class of lipids in asexual blood stage P.

falciparum. The blood stage parasites have a high abundance of the structural sphingolipid, sphingomyelin (approx. 10-15%) and the unexpected presence of dihydrosphingomyelin (dhSM) (approx. 1%) [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. P. falciparum is able to induce the formation of an extensive network of tubovesicular membranes (TVM) extending from the PVM to the periphery of the host cell, necessary for nutrient uptake (Lauer et al., 1997). The parasite exports its own sphingomyelin synthase into the host erythrocyte to synthesize sphingomyelin necessary for the development of the tubovesicular membranes (Elmendorf and Haldar, 1994;[START_REF] Haldar | Sphingolipid synthesis and membrane formation by Plasmodium[END_REF]. Other than this, P.

falciparum has been shown to have sphingomyelin synthase activity in its Golgi apparatus [START_REF] Lauer | Sphingolipid synthesis as a target for chemotherapy against malaria parasites[END_REF].

Cholesterol and cholesteryl esters

Cholesterol exists either as a ubiquitous component of membranes or in fatty acid esterified form, cholesteryl ester within lipid storage droplets of the parasites. Apicomplexan parasites are auxotrophic for cholesterol and can compensate via copious salvage of cholesterol directly from the host (Coppens, 2013). As determined by filipin based staining, cholesterol in Toxoplasma tachyzoites is concentrated within plasma membrane, apical organelles and rhoptries [START_REF] Coppens | Toxoplasma gondii exploits host low-density lipoprotein receptormediated endocytosis for cholesterol acquisition[END_REF]. Toxoplasma exploits the host low-density lipoproteins (LDL)for direct uptake of exogenous cholesterol. Consistent with the exogenous cholesterol uptake, impaired de novo cholesterol biosynthesis within host cells, does not affect the parasite growth. A membrane bound efflux pump in the host, P-glycoprotein participates in the transport of host-derived cholesterol towards the intracellular parasite [START_REF] Bottova | Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii[END_REF].

Toxoplasma stores excess fatty acids as palmitate and oleate by their esterification to cholesterol the form of palmitate and oleate containing cholesteryl esters (Coppens, 2013). Toxoplasma genome encodes for two enzymes that are involved in cholesteryl ester biosynthesis, acyl CoA: cholesterol acytransferase1 (ACAT1) and acyl CoA acyltransferase2 (ACAT2). Genetic ablation of each the individual ACATs impairs parasite growth due to reduced levels of cholesteryl esters and consequent increase in the amount of membrane cholesterol. The double disruption of ACAT1 an ACAT2 is lethal to Toxoplasma tachyzoites [START_REF] Lige | Characterization of a second sterol-esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite[END_REF].

Liver-stage P. falciparum obtains its cholesterol directly from host mevalonate pathway and via LDL and HDL (exogenous sources) [START_REF] Grellier | Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells[END_REF][START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF]. The proportion of cholesterol fluctuates between 47% in uninfected RBCs to a decrease with 19% in RBCs infected with trophozoites and then increase again to 29% when the infected RBCs mature to sexual stage gametocytes [START_REF] Tran | Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum[END_REF]. Despite an early report on the absence of cholesteryl esters from P. falciparum [START_REF] Nawabi | Neutral-lipid analysis reveals elevation of acylglycerols and lack of cholesterol esters in Plasmodium falciparum-infected erythrocytes[END_REF], the parasite does store cholesterol and excess fatty acids in the form of cholesteryl esters [START_REF] Tran | A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite[END_REF].

Phosphatidic acid

Phosphatidic acid (PA) is the simplest glycerophospholipid bearing a negative charge. This anionic lipid has a phosphomonoester head group. PA acts as a central precursor molecule for the biosynthesis of various other glycerolipids including triglycerides. The cone shaped symmetry of this peculiar phospholipid results in a negative membrane curvature thereby allowing its participation in key membrane remodeling events like fission and fusion (Bullen and Soldati-Favre, 2016;[START_REF] Tanguy | Phosphatidic acid: From pleiotropic functions to neuronal pathology[END_REF]. Phosphatidic acid comprises 1% of the total polar lipid composition in T. gondii (Welti et al., 2007).

Biogenesis of phosphatidic acid: Reference to apicomplexan enzymes

In various eukaryotes, different species of phosphatidic acid are generated by at least four different pathways. The main pathway contributing to PA synthesis begins with the glycolytic intermediate glycerol-3-phosphate and involves two sequential reactions with acyltransferases-acyl CoA:

glycerol-3-phosphate acyltransferase (GPAT) and acyl CoA: lysophosphatidic acid acyltransferase (LPAAT) [START_REF] Athenstaedt | Phosphatidic acid, a key intermediate in lipid metabolism[END_REF]. The second pathway also uses acyltransferases however with the involvement of a different substrate i.e. dihydroxyacetone phosphate (DHAP). The third pathway employs the enzyme phospholipase D, which hydrolyses phospholipids like PC to generate phosphatidic acid. Lastly, under certain conditions, diacylglycerol kinases (DGKs) can catalyze the formation of PA in an ATP dependent manner from the substrate diacylglycerol (DAG).

In T. gondii, a complete de novo pathway of PA biosynthesis localizing to the apicoplast has been identified (Amiar et al., 2016(Amiar et al., , 2019)). Briefly, phosphatidic acid is synthesized within apicoplast by the sequential esterification of fatty acyl-CoA onto a glycerol-3-phosphate backbone by glycerol-3-phosphate acyltransferase (AGPAT/ATS1) forming lysophosphatidic acid (LPA) and then by an acylglycerol-3-phosphate acyltransferase (AGPAT/ATS2) forming PA from LPA. The parasite has also been shown to uptake BODIPY-phosphatidic acid from the extracellular environment and the host [START_REF] Charron | Host cells: Mobilizable lipid resources for the intracellular parasite Toxoplasma gondii[END_REF].

Plasmodium on contrary has an incompletely mapped apicoplast PA biosynthetic pathway, with two known enzymes sufficient to catalyze conversion of acyl-ACPs and dihydroxyacetone phosphate (DHAP) into the intermediate lysophosphatidic acid [START_REF] Lindner | Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development[END_REF]Shears et al., 2017). It is believed that in Plasmodium, the apicoplast generates only lysophosphatidic acid which is fully acylated to generate phosphatidic acid within the parasite ER. The table below (Table 1)

represents a list of all the enzymes that have been characterized to be involved in phosphatidic acid biosynthetic pathway in apicomplexan parasites Toxoplasma gondii and Plasmodium sp.

TgATS1

-apicoplast localized -indispensable for toxoplasma tachyzoites -plant like glycerol-3-phosphate acyltransferase that generates lysophosphatidic acid, the precursor for phosphatidic acid biosynthesis (Amiar et al., 2016) TgATS2 -apicoplast localized -catalyzes the esterification of an activated FA (i.e. acyl-CoA or acyl-ACP) onto LPA to make PA (Amiar et al., 2019)

TgDGK1

-DGK phosphorylates phosphoinositide phospholipase C (PI-PLC)-derived diacylglycerol (DAG) to form PA, at the parasite plasma membrane (Bullen et al., 2016)

TgDGK2

-secreted into parasite parasitophorous vacuolar space -produces PA in the parasitophorous vacuolar space which further acts as an intrinsic signal for natural parasite egress [START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF] Pf apiG3PAT -dispensable during the blood stage of P. falciparum -an apicoplast localized glycerol-3-phosphate acyltransferase (G3PAT)

-likely catalyzes the second step in apicoplast localized lysophosphatidic acid biosynthesis (Shears et al., 2017)

PyapiG3PDH

-essential for the late liver stage development of Plasmodium yoelii -a glycerol-3-phosphate dehydrogenase (G3PDH) that likely catalyzes the first step of PA biosynthesis via conversion of glycolytic intermediate [START_REF] Lindner | Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development[END_REF] dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate using NADPH as an electron donor

PyapiG3PAT

-essential for late liver stage development of P. yoelii -this glycerol-3-phosphate acyltransferase localizes to the parasite apicoplast -likely catalyzes the formation of lysophosphatidic acid, the immediate precursor of PA [START_REF] Lindner | Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development[END_REF] Table 1: Apicomplexan enzymes involved in phosphatidic acid biosynthesis Phosphatidic acid at the forefront of the biological armor of parasites: Key roles in parasite biology and lipid metabolism a) Membrane biogenesis: In eukaryotes, phosphatidic acid can be dephosphorylated by an enzyme phosphatidate phosphatase (PAP) in order to yield diacylglycerol (DAG), which serves as a precursor for the formation of major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine via Kennedy pathway [START_REF] Athenstaedt | Phosphatidic acid, a key intermediate in lipid metabolism[END_REF]. Toxoplasma apicoplast expresses a plant like acyltransferase called ATS1. TgATS1 has been shown to catalyze the incorporation of FASII derived fatty acids into lysophosphatidic acid (LPA), an immediate precursor of PA (Amiar et al., 2016). Genetic disruption of TgATS1 resulted in reduced incorporation of FASII-synthesized fatty acids, specifically myristate (C14:0) and palmitate (C16:0) into PA and downstream phospholipids (PC, PE and PI) and consequently a severe defect in intracellular parasite replication and survival.

b) Lipid signaling modulating parasite invasion, motility and egress: Phosphatidic acid (PA) and diacylglycerol (DAG) have been implicated as important lipid mediators involved in key signaling events in mammalian cells (Bullen and Soldati-Favre, 2016). Several studies in the field of apicomplexa support the involvement of PA in establishment of infection inside the host cells.

In P. falciparum liver stage, CelTOS, a protein involved in the traversal of sporozoites across hepatocytes, has been shown to do so by binding and disruption of PA-enriched membranes (Jimah et al., 2016). Glideosome associated connector (GAC) is an actin binding protein which controls the gliding motility and invasion in Toxoplasma. GAC links adhesin-MIC2 to parasite actomyosin system while interacting with phosphatidic acid in the parasite plasma membrane via its PH domain (pleckstrin homology-PH) (Jacot et al., 2016).

The maintenance of parasite's intra cellular lifestyle is dependent on coordinated invasion and egress events relying on spatially controlled secretion of apical organelles called micronemes (Bullen et al., 2016). Bullen et al, showed that a delicate balance between PA and DAG governed by a Toxoplasma diacylglycerol kinase-1 (DGK1) is the basis for regulated event of microneme secretion. Genetic disruption of TgDGK-1 localized at the parasite periphery resulted in parasite cell death. The impact of PA generated by TgDGK-1in microneme secretion is mediated by contact with a microneme surface protein bearing pleckstrin homology domain-APH1. Thus, in Toxoplasma PA acts as the lipid mediator of key signaling event involving regulated exocytosis by the recruitment of micronemes via specific effector proteins.

Phosphatidic acid generated by the action of a second diacylglycerol kinase DGK2, is involved in the egress event of parasite infectivity within its host. This enzyme is secreted into the parasitophorous vacuole where it likely generates PA which in turn acts as an intrinsic signal facilitating parasite egress upstream of an atypical guanylate cyclase (GC) [START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF].

c) Cytokinesis and parasite cell division

Phosphatidic acid and its immediate precursor lysophosphatidic acid balance effectively allowing normal parasite division. A dynamin related protein TgDrpC participates during T. gondii endodyogeny which in turn relies on the local LPA/PA balance generating appropriate membrane curvature, thereby assisting in cytokinesis of daughter cells (Amiar et al., 2019).

d) Lipid storage

The unprecedented role of PA in storage is yet to be assessed in apicomplexan parasites. One of the branches of PA metabolism leads to synthesis of triglycerides in various eukaryotic cells [START_REF] Athenstaedt | Phosphatidic acid, a key intermediate in lipid metabolism[END_REF]. The hydrolysis of PA generating DAG molecules acts as the substrate for acyltransferases resulting in the generation of triacylglycerols which are major constituents of lipid storage bodies. et al., 2016) and cell division process by maintaining a balance with its own precursor (LPA) (Amiar et al., 2019) (b). This dynamic lipid also acts as signaling molecule participating in keys parasite pathogenicity events like invasion and egress (Bullen et al., 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF]. PA is also channeled towards lipid storage pathway ( [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]this thesis).

Triacylglycerols

Biochemically, triacyclglycerols (TAGs) are highly hydrophobic, inert and energetically dense glycerolipid molecules. The TAGs are stored within the distinct cytoplasmic organelles called lipid droplets surrounded by a monolayer of phospholipids. The fatty acids released via TAG hydrolysis serve either for structural purpose in phospholipid biosynthesis or are channeled towards energy metabolism via β-oxidation.

Initial studies on lipid species of P. falciparum infected RBCs suggested that the asexual blood stages were able to accumulate a high amount of DAG and TAG [START_REF] Nawabi | Neutral-lipid analysis reveals elevation of acylglycerols and lack of cholesterol esters in Plasmodium falciparum-infected erythrocytes[END_REF]. Metabolic labelling studies using palmitic acid (C16:0) and oleic acid (C18:1) revealed a striking accumulation of TAGs from trophozoite to schizonts in P. falciparum asexual life cycle [START_REF] Lk -Palacpac | Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes[END_REF]. TAGs have also been reported to be involved in the maturation of asexual stage parasites. The use of orlistat, an inhibitor of TAG hydrolysis, on P. falciparum asexual life cycle arrested the parasites soon after the onset of merozoite formation [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF].

In various eukaryotes, PA and DAG are two main substrates involved in glycerolipid biosynthesis pathway contributing to TAG biosynthesis. The apicomplexan enzymes involved in this pathway are discussed below: a) Phosphatidic acid phosphatase: The phosphatidic acid species committed to TAG pathway is first dephosphorylated to generate DAG via a phosphatidic acid phosphatase. The genome of Toxoplasma and P. falciparum have been annotated to encode three and two phosphatidic acid phosphatases respectively (Bullen et al., 2016). One of the PAPs, called PAP2-like protein, localizes to the cytoplasm of Toxoplasma in the form of vesicular sutures. This PAP2-like protein was shown to be indispensable for parasite growth probably due to this is its less significant contribution to PA/DAG balance. The Plasmodium homologue, PfPAP2 has been shown to contribute to delicate PA and DAG balance necessary for maintaining complete lytic cycle via parasite egress [START_REF] Kumar Sah | Phosphatidic acid homeostasis regulated by a type-2 phosphatidic acid phosphatase represents a novel druggable target in malaria intervention[END_REF]. Another PAP studied, has been shown to localizes towards the IMC sutures of T. gondii [START_REF] Chen | Novel components of the toxoplasma inner membrane complex revealed by BioID[END_REF]. In various other eukaryotic cells, most of the phosphatase activity leading to TAG biosynthesis resides in cytoplasmic proteins called 'lipins' (Reue and Wang, 2019). Lipins are evolutionarily conserved proteins regulating lipid metabolism in different cell types [START_REF] Csaki | Lipins, lipinopathies, and the modulation of cellular lipid storage and signaling[END_REF]. These phosphatidic acid phosphatases catalyze the penultimate step of the glycerol-3-phosphate pathway via a Mg 2+ dependent dephosphorylation of PA to DAG, implicating further in triglyceride and phospholipid biosynthesis. Sometimes these key regulator proteins have also been shown to have transcriptional co activator activity monitoring the expression of various genes involved in lipid metabolism. Both Toxoplasma and Plasmodium have one genome-annotated lipin

which are yet to be characterized. In Toxoplasma, lipin has been localized towards parasite cytoplasm, without any further characterization with respect to the glycerolipid biosynthesis pathways (Bullen et al., 2016).

b) Acyl CoA: diacylglycerol acyltransferase (DGAT): In the final step of the glycerolipid biosynthesis pathway, the DAG species are acylated to fatty acids with the help of enzymes called acyl: coA diacylglycerol acyltransferase. The biosynthetic machinery for TAG formation has not been characterized completely in apicomplexa. However, there are evidence for existence of DGAT homolog named TgDGAT. TgDGAT, localizing towards the parasite cortical and perinuclear endoplasmic reticulum preferentially incorporates palmitic acid (C16:0) into TAGs [START_REF] Quittnat | On the biogenesis of lipid bodies in ancient eukaryotes: Synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme[END_REF]. This suggests that TAG biosynthesis in apicomplexa mirrors other eukaryotes, involving the substrates PA and then DAG. Oleic acid (C18:1) appears to be one of the major constituents of TAGs in Toxoplasma. Feeding the parasites with exogenous oleic acid in a concentration dependent manner (0.2 and 0.4 mM) increases the transcriptional activity of TgDGAT (by up to 1.5fold) allowing the influx of oleate to be channeled towards acylglycerols (Nolan, Romano and Coppens, 2017). However, in case of excess oleic acid (0.5mM and above), the increased TgDGAT activity is unable to protect the parasite from lipotoxic response, resulting in an irreversible replication arrest [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. The vulnerability of Toxoplasma tachyzoites and bradyzoites to a chemical inhibitor (T863) of DGAT highlighted the importance of neutral lipid synthesis and storage for avoiding lipotoxicity within parasites. T863-treated tachyzoites show abnormal ER-derived membranous structures that are stockpiled in the cytoplasm, likely impeding normal endodyogeny. Dual addition of oleic acid (0.5 mM) and T863 synergistically deteorates parasite growth. The excess fatty acids unable to be transformed towards storage TAGs due to TgDGAT inactivity are likely channeled towards phospholipid biosynthesis leading to excess membrane phenotype in the presence of T863. The treatment of Toxoplasma bradyzoites with T863 lead to misshapen cysts and reduced lipid droplets (LD) in bradyzoites, although sensitivity occurs at concentrations higher than those observed for tachyzoites.

FATTY ACID METBOLISM IN APICOMPLEXA

Fatty acids are the glycerolipid structural building blocks, which can also serve as energy storage molecules and moieties for the post-translational modification of specific groups of proteins [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]. Amongst apicomplexan parasites, T. gondii employs three major de novo metabolic pathways for coping the pressure of continuous need of fatty acid biosynthesis. These include the apicoplast-resident prokaryotic type-II fatty acid biosynthesis pathway, the ER-based fatty acid elongation pathway and the cytosolic eukaryotic type-I fatty acid synthase (Mazumdar and Striepen, 2007). Despite such a complex de novo FA synthesis network, the parasites also undertake copious salvage of fatty acids directly from the host.

De novo type II fatty acid biosynthesis pathway

The fundamental process of fatty acid biosynthesis is based on of sequential extension of an alkanoic chain, two carbons at a time, by a series of decarboxylative condensation reactions [START_REF] Chirala | Structure and function of animal fatty acid synthase[END_REF]. In general, the process of FA biosynthesis begins with carboxylation of acetyl-coA to malonyl-coA. This process is generally initiated with the carboxylation of acetyl-CoA to yield malonyl-CoA [START_REF] Smith | Structural and functional organization of the animal fatty acid synthase[END_REF]. Next, initiation of condensation reactions begins with translocation of acetyl and malonyl moieties to the malonate group of malonyl-com, which is further is transferred to the phosphopantetheine prosthetic group of a small, acidic protein, called the acyl carrier protein (ACP). This is followed by a series of reactions involving reduction, dehydration and reduction again yielding an acyl-ACP. The elongation of the chain occurs by condensing another malonyl-ACP with the acyl-ACP and repeating the reaction cycle.

In apicomplexan parasites, T. gondii and P. falciparum, the fundamental FASII pathway occurs within apicoplast. The apicoplast FASII initiation is reliant on glycolytic intermediate phosphoenol pyruvate (PEP). Transport of these glycolytic intermediates into apicoplast is dependent on antiporters that move phosphorylated carbon compounds across membranes in exchange for inorganic phosphate. Plasmodium is known to have two triose phosphate transporters each one in the outer (PfoTPT) and inner (PfiTPT) membrane of the apicoplast [START_REF] Mullin | Membrane transporters in the relict plastid of malaria parasites[END_REF].

Toxoplasma on the other hand has a single phosphate transporter TgAPT, which although localized to apicoplast membranes has not been delineated to a specific membrane of the organelle. TgAPT is essential for parasite growth and development [START_REF] Brooks | The Toxoplasma Apicoplast Phosphate Translocator Links Cytosolic and Apicoplast Metabolism and Is Essential for Parasite Survival[END_REF]. Heterologous complementation of yeast with TgAPT functionally validated it as a transporter specific for triose phosphates (ATP, NADPH), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP).

Pyruvate kinase (PK) embarks the second step and the beginning of preparation phase of FASII by catalytically converting PEP to pyruvate and releasing ATP as a by-product. The genomes of both P. falciparum and T. gondii encode two pyruvate kinases PYKI and PYKII, out of which PKI shares strong homology cytoplasmic glycolytic enzyme. The PYKII, localizes to apicoplast in P. falciparum [START_REF] Maeda | Pyruvate kinase type-II isozyme in Plasmodium falciparum localizes to the apicoplast[END_REF] whereas its Toxoplasma homolog localizes to both mitochondria and apicoplast. In Toxoplasma PYKI is refractory to genetic deletion because of its important role in the incorporation of glucose-or glutamine-derived carbon into glycolysis, TCA cycle, amino acids, and fatty acid synthesis pathways [START_REF] Xia | Pyruvate homeostasis as a determinant of parasite growth and metabolic plasticity in toxoplasma gondii[END_REF]. TgPYKII shows unusual substrate specificity of GDP over ADP and is not essential for parasite growth [START_REF] Saito | A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion[END_REF][START_REF] Xia | Pyruvate homeostasis as a determinant of parasite growth and metabolic plasticity in toxoplasma gondii[END_REF]. Double disruption of both the pyruvate kinases was lethal to Toxoplasma tachyzoites. The next step is conversion of pyruvate to acetyl coA, a reaction releasing carbon dioxide, catalyzed by enzyme complex called pyruvate dehydrogenase (PDH). Both P. falciparum and T. gondii genomes encode a PDH localizing towards the apicoplast [START_REF] Foth | The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast[END_REF]Fleige et al., 2007). The mitochondrial PDH activity is bypassed by the presence of branched chain ketoacid dehydrogenase (BCKDH) complex in T. gondii as well as P. berghei [START_REF] Oppenheim | BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei[END_REF].

The acetyl coA and bicarbonate are condensed to yield malonyl coA using ATP and biotin as a cofactor. This reaction is catalyzed by enzyme called acetyl coA carboxylase (ACC) which exists as a single multi-functional enzyme in P. falciparum and two isoforms T. gondii. TgACC1 is localized to the apicoplast functioning for FASII whereas the second ACC is present in the cytosol possibly fueling initial step of FASI pathway [START_REF] Jelenska | Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii[END_REF].

The next step involves catalytic transfer of Malonyl coA to ACP, aided by malonyl coA: ACP transacylase (FabD), resulting in formation of malonyl-ACP [START_REF] Shears | Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts[END_REF].

Acyl carrier protein (ACP) is the central carrier protein that binds to fatty acids and their precursors via a phosphopantetheine prosthetic group. ACP essentially participates in transferring substrates between different enzymes of the FASII pathway. With no genetic studies done on PfACP, the TgACP has been shown to be refractory to deletion. Conditional knockdown studies on TgACP show loss of apicoplast which finally leads to arrest of growth of tachyzoites (Mazumdar et al., 2006). Studies using the conditional null mutant of TgACP determined for the first time that the sole carbon source for apicoplast FASII was glucose rather than acetate and the apicoplast derived fatty acids are usually of the chain length of 12-16 carbon atoms (Mazumdar et al., 2006;Ramakrishnan et al., 2012). This research therefore affirmed the use of labelled glucose to track the direct fatty acid derivatives of FASII pathway.

Catalyzing the last reaction of the FASII initiation phase is β-Ketoacyl-ACP synthase III (FabH).

The last condensation reaction involves two 2-carbon substrates acetyl coA and malonyl-ACP, generating a 4-carbon product acetoacetyl-ACP (β-Ketoacyl-ACP) which is further used as a scaffold for the elongation phase of FASII. This reaction is catalyzed by β-Ketoacyl-ACP synthaseI/II , also known as FabB/F. FabH is also a β-Ketoacyl-ACP synthase III that is able to bypass the reaction catalyzed by FabB/F [START_REF] Shears | Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts[END_REF].This reaction releases CO2 and coA, as by-products.

The elongation phase of FASII pathway involves a series of three sequential enzymatic reactions increasing the length of carbon skeleton by 2-carbons at one time. β-Ketoacyl-ACP is reduced by β-ketoacyl: ACP reductase (FabG) to form β-hydroxyacyl-ACP using NADPH as an electron donor. This product is then dehydrated by β-hydroxyacyl-ACP dehydratase (FabZ) to form α, βtrans enoyl-ACP. FabZ and FabB/F KO studies revealed the dispensability of FASII for Plasmodium blood stages. FabZ however has been shown to be essential for the parasite's transition from the liver to blood due to its indispensability for late stage liver development (Vaughan et al., 2009). This is further reduced to butyryl-ACP by the action of enoyl-ACP reductase (FabI). Genetic depletion studies of FabI provided the first evidence of FASII indispensability for only liver stages of the parasite development. This piece of data also created the barrier between metabolic requirements for different stages of Plasmodium development. The dispensability of blood stage FASII suggested the parasite's dependence on the host for its metabolic needs rather than complete reliance on apicoplast FASII (Yu et al., 2008). Further studies on FabI during the mosquito stage parasite life cycle that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst (van Schaijk et al., 2014).

Morphological analysis using combined light and electron microscopy techniques confirmed the essentially of FASII pathway within Toxoplasma tachyzoites (Ramakrishnan et al., 2012;Amiar et al., 2016). Toxoplasma tachyzoites lacking FASII activity were unable to complete the process of cytokinesis and eventually gave rise to daughter cells tethered to one another at their basal ends [START_REF] Martins-Duarte | Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii[END_REF]. The phenotype arose due to inability of the parasite to supply fatty acids essential for membrane biogenesis. At the basal ends of these parasites the pellicle formation was incomplete due to the incomplete biogenesis of the IMC and the plasma membrane of the parasite. Exogenous fatty acids were able to only partially revert the tethered phenotype of daughter cells, thereby confirming the essential contribution of FASII derived fatty acids towards membrane biogenesis.

Another functional pathway belonging to the apicoplast is biosynthesis of short chain fatty acid called 'lipoic acid'. Lipoic acid is an organosulfur-derivative of octanoic acid which acts as an essential cofactor for several multi-subunit enzymes esp. the a-keto acid dehydrogenase complexes [START_REF] Wrenger | The human malaria parasite Plasmodium falciparum has distinct organelle-specific lipoylation pathways[END_REF]. One of the short chain fatty acid products of FASII, octanoyl-ACP, is channeled towards lipoic acid biosynthesis pathway. The lipoic acid is formed by catalytic ligation of S-adenosylmethionine (SAM) with octanoyl-ACP. Lipoic acid biosynthesis in the malarial parasites has been differentially compartmentalized. The enzymes involved in this pathway include lipoic acid synthase, lipoyl-ACP: protein N-lipoyl transferase both localizing to apicoplast and lipoic acid protein ligase localizing to the mitochondrion of the parasite. ACPknockdown parasites are impaired in lipoylation of the apicoplast PDH. Basically, the apicoplast produces lipoic acid to continue the essential FASII via a functional PDH acting as the only source of acetyl coA in the organelle.

Fatty acid elongation pathway

The second pathway contributing to FA biosynthesis in apicomplexa is the elongation machinery localized to the endoplasmic reticulum of the parasite. The FASII and elongation pathway are nonredundant in terms of their functional capacities of maintaining FA bulk synthesis within the apicomplexan parasites (Ramakrishnan et al., 2012).

T. gondii acetyl coA synthetase enzyme provides acetyl coA for the initial step of the fatty acid elongation pathway (Dubois et al., 2018). However, disruption of TgACS has a minor effect on the global fatty acid composition and subsequently parasite intracellular replication, likely due to the metabolic flux changes induced to compensate for its loss.

The ER -based elongation machinery in T. gondii comprises of three elongases, two reductases, and a dehydratase (Ramakrishnan et al., 2012). The loss of three elongases ELO-A, ELO-B and ELO-C did not impede parasite growth suggesting their redundant function. This could also mean that intracellular parasites were able to compensate for the loss of the elongases by scavenging FAs directly from the host cell. On contrary, the parasite mutants deficient in the dehydratase (DEH) and enoyl reductase (ECR) enzymes of the fatty acid elongation pathway exhibited severe growth defect [START_REF] Ramakrishnan | The intracellular parasite Toxoplasma gondiidepends on the synthesis of longchain and very long-chain unsaturated fatty acids not supplied by the host cell[END_REF]. The incorporation of 14 C-acetate into fatty acids was dramatically decreased in iΔDEH parasites. The loss of DEH activity resulted in depletion of synthesis of unsaturated long (LFCA) and very long chain fatty acids (VLFCA) (C18:1 to C26:1) in phosphatidylethanolamine and phosphatidylinositol species. The growth defect of iΔDEH parasites in the presence of ATc was rescued by supplementation of the medium with a mixture of saturated-and monounsaturated LCFA and VLCFA.

During the FA elongation pathway, the parasites are first desaturated and then further elongated with the help of elongase complexes present in the parasite (Ramakrishnan et al., 2012). The existence of monounsaturated and polyunsaturated FAs in the parasites can be owed to the existence of enzyme desaturases and also the fatty acids scavenged directly from the host [START_REF] Ramakrishnan | Lipid synthesis in protozoan parasites: A comparison between kinetoplastids and apicomplexans[END_REF]. Stearoyl coA desaturases (SCD) are enzymes catalyzing the introduction of a single double bond in a saturated FA backbone thereby generating a monounsaturated FA. There is both genetic and biochemical evidence for the presence of a ∆9desaturase domain containing SCD in apicomplexan parasites P. falciparum and T. gondii [START_REF] Gratraud | Oleic acid biosynthesis in Plasmodium falciparum: Characterization of the stearoyl-CoA desaturase and investigation as a potential therapeutic target[END_REF]. In P. falciparum, the SCD localizes towards the parasite ER and is necessary for maintaining the intra-erythrocytic developmental cycle by catalyzing the conversion of stearic acid to oleic acid. The Toxoplasma cell line over expressing the desaturase TgSCD is able to increase the levels of palmitoleic acid and oleic acid consistent with its function of a desaturase [START_REF] Hao | Identification and characterization of stearoyl-CoA desaturase in Toxoplasma gondii[END_REF].

The concept of 'Patchwork lipids' in Toxoplasma gondii: FAs derived from host and de novo synthesis

The survival of Toxoplasma within its host is predominantly dependent on lipids, which play an essential role by regulating metabolic flux. The demand for lipids in these parasites relies on an essential combination or 'patchwork' of fatty acids (FA) synthesized de novo by the apicoplast FASII pathway and/or by directly salvaging from the host (Mazumdar et al., 2006;Ramakrishnan et al., 2012;Amiar et al., 2016;[START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF][START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. Mechanisms allowing the parasite to strike a balance between two different sources of fatty acids remain largely unknown.

However, the advent of stable isotope labelling using precursors like U-13 C-glucose or U- 13 Cacetate has allowed determination of source of these fatty acid fluxes. Fatty acids showing label incorporation with U-13 C-glucose are de novo synthesized fatty acids via FASII in apicoplast whereas the incorporation of U-13 C-acetate to fatty acids determines the elongation of fatty acids in cytosol (Ramakrishnan et al., 2012[START_REF] Ramakrishnan | The intracellular parasite Toxoplasma gondiidepends on the synthesis of longchain and very long-chain unsaturated fatty acids not supplied by the host cell[END_REF]Amiar et al., 2016). The FA label incorporation is technically distinguished by the shift of mass using gas-chromatography coupled mass spectrometry (GC-MS).

FA synthesis and flux from within the parasite: Experimental evidences

Metabolic labelling studies with U-13 C-glucose have shown that intracellular tachyzoites are capable of synthesizing a range of long and very long chain fatty acids (C14:0-26:1) (Ramakrishnan et al., 2012). 13 C-Glc based labelling of the ACP knockdown parasites showed a reduction in the FASII activity marked by 80% reduction of the label incorporation into myristic (C14:0) and palmitic acid (C16:0). Genetic ablation of three independent elongases provided insights into the FA fluxes dealt by the FA elongation pathway of the parasite. ELO-A is required for efficient elongation of C16:0 or C16:1 to longer species. ELO-B is largely responsible for the elongation of C18:1/C20:1 to C22:1, whereas the primary role of ELO-C is to elongate fatty acids from C22:1 to C26:1. FASII derived fatty acids are directly incorporated into lipids used for building membranes during the intracellular development of parasites. Use of a FASII inhibitor, Triclosan on Toxoplasma tachyzoites resulted in development of a cytokinesis defect due to insufficient membrane biogenesis. The consequent tethered daughter cell phenotype of these parasites was partially rescued by addition of exogenous fatty acids C14:0, C16:0, C18:1, C22:1. Supplementation of C14:0 alone and in the combination with C16:0 was most successfully able to revert the tethered phenotype, suggesting the importance of these FASII derived fatty acids in parasite membrane biogenesis [START_REF] Martins-Duarte | Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii[END_REF].

Disruption of ATS1 is lethal to Toxoplasma and results in major significant decrease in incorporation of the FASII-generated fatty acid C14:0 (myristate) into bulk synthesis of major phospholipid classes found throughout the parasite membranes. This adverts the importance of apicoplast derived fatty acid flux within the parasite, which cannot always be bypassed by scavenging from the host (Amiar et al., 2016).

Stable isotope labeling using 13 C-acetate combined with mass spectrometry-based lipidomic analyses, showed that TgACS is involved in providing acetyl-CoA for the fatty elongation pathway in the ER to generate very long fatty acids C18:0, C18:1, C20:0, C20:1, C22:0, C24:0 (Dubois et al., 2018).

FA flux from the host: Experimental evidences

The apicomplexan parasites are auxotrophic for certain metabolites and compensate their demand by scavenging directly from the host and their environment. One of the important metabolites scavenged directly from the host include lipids and fatty acids (Coppens, 2013). The intraerythrocytic malarial parasite blood stages are dependent mostly on the host for their fatty acid demand whereas the sexual life cycle of the malarial parasite within mosquito midgut relies mostly on the de novo FA biosynthetic pathway (van Schaijk et al., 2014).

Extracellular fatty acids myristic acid (C14:0) and palmitic acid (C16:0) get incorporated to glycosylphosphatidylinositol (GPI) anchors on the surface antigens of T. gondii [START_REF] Tomavo | Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens[END_REF]. A remarkable study from Charron and Sibley, provided insights into mobilization of host lipids from various cellular compartments into lipid body and selective endomembrane compartments within the parasite [START_REF] Charron | Host cells: Mobilizable lipid resources for the intracellular parasite Toxoplasma gondii[END_REF]. This study involved the use using radioisotope and fluorescently labelled lipid precursors for host and parasite labelling and its subsequent detection and analysis by confocal microscopy. NBD-cholesterol was mobilized from pre-labelled host perinuclear compartments to distinct punctate localizations within intracellular parasites. BODIPY-PtdCho (Phosphatidylcholine)was able to move to plasma membrane and distinct punctate localizations within the host and their infected parasites. However, parasites infected after pre-labelling the host were not able to accrue BODIPY-PtdCho, thereby suggesting that direct uptake of PtdCho from the host was unlikely. The uptake of BODIPY-PA (Phosphatidic acid) and fatty acid C4-BODIPY-C9 was concentrated towards large puncta in parasites as well as in a membrane separating the parasite from pre labelled host. The same study also showed that parasites are able to metabolize host-derived neutral lipids including NBDcholesterol and the BODIPY-conjugated C12 fatty acid. These parasites are also able to uptake lipids in the form of neutral lipid stores directly from the host (Nolan, Romano and Coppens, 2017).

Alongside, acquisition of fatty acids and potentially direct phospholipids from the host, T. gondii is also able to scavenge the lipid precursors serine, ethanolamine, and choline from its environment. These polar headgroups are used by the parasite for the synthesis of its major lipids including phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF].

Toxoplasma is auxotrophic for cholesterol biosynthesis and is completely reliant for the same on its host. The parasite exploits host cholesterol complexed to low density lipoproteins for cholesteryl ester (CE) synthesis and storage within lipid bodies [START_REF] Nishikawa | Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma[END_REF]. The CE biosynthesis is aided by endoplasmic-reticulum localized acyl CoA: cholesterol acyltransferase (ACAT). Amongst the various radiolabeled fatty acid sources fed to extracellular parasites, palmitic acid (C16:0) was most readily incorporated into CE. Similarly, excess oleate (C18:1) and palmitate (C16:1) in the extracellular medium resulted in 3-4-fold increase in CE biosynthesis within the parasite, which further corroborated with the increased mRNA ACAT expression.

Lipoic acid, an essential co-factor of α-keto dehydrogenases in apicoplast (PDH) and mitochondria (BCKDH) is scavenged from external host environment despite its de novo synthesis within apicoplast. This scavenged lipoic acid is specifically channeled towards mitochondria thereby probating a potential link between mitochondria and parasitophorous vacuole membrane for scavenging from the host directly [START_REF] Crawford | Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast[END_REF]. Other abbreviations used: TgACC1, acetyl CoA carboxylase 1; ACP, acyl carrier protein; PDH, pyruvate dehydrogenase; PK, pyruvate kinase; TPT, triose phosphate transporter; PEP, phosphoenol pyruvate (adapted from [START_REF] Ramakrishnan | The intracellular parasite Toxoplasma gondiidepends on the synthesis of longchain and very long-chain unsaturated fatty acids not supplied by the host cell[END_REF].

Acquiring the fats: Potential methods of lipid scavenging by Toxoplasma

The parasite mediates the essential lipid uptake via several routes depending upon the substrate of interest:

a) via the intravacuolar network Intravacuolar network (IVN) comprises an extensive network of thin tubules and vesicles that fill the lumen of parasitophorous vacuole [START_REF] Caffaro | Evidence for host cells as the major contributor of lipids in the intravacuolar network of toxoplasma-infected cells[END_REF].

Interestingly, a Toxoplasma mutant lacking the capacity to form IVN (∆gra2∆gra6) was unable to embark the process of lipid uptake from the host (Nolan, Romano and Coppens, 2017). Also, parasites lacking an IVN localized phospholipase exhibit reduced host lipid scavenging capacity. The involvement of a lipid hydrolyzing enzyme in the parasite vacuolar space suggests that the host lipids are recycled right at the point of entry into the parasites.

b) via physical association of the PVM and host cell organelles allowing mobilization of lipids.

Host cell mitochondria and endocytic machinery arising from Golgi complex are actively shown to be around PVM post Toxoplasma infection, participating in lipid metabolic homeostasis within the parasite [START_REF] Coppens | Toxoplasma gondii exploits host low-density lipoprotein receptormediated endocytosis for cholesterol acquisition[END_REF][START_REF] Pernas | Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response[END_REF][START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. In T. gondii type I and type III strains a parasite protein, MAF1 (mitochondrion associated factor), is necessary for host mitochondrial association with the parasite vacuole [START_REF] Pernas | Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response[END_REF].

c) uptake of host lipids in an ATP-dependent process

Toxoplasma has a family of ATP-binding cassette (ABC) G family transporter proteins that participate in the uptake of host lipids and the expulsion of major lipids from the parasite (Ehrenman et al., 2010). One of these ACBGs, TgACBG107 is associated with the vesicles in the PV and plasma membrane participating in the cholesterol import from the host. Four out of the several ABCGs, TgABCG77, TgABCG84, TgABCG89 and TgABCG87 are selectively involved in cholesterol and phosphatidylcholine export.

d) carrier proteins mediated transfer

Fatty acyl-coA esters are amphipathic molecules that can disrupt the membranes they traverse through. These detergent-like molecules need carrier proteins for transport across various endomembrane compartments. Acyl coA binding protein (ACBP) is one such carrier protein that binds fatty acyl coA esters and transports them across various cellular compartments [START_REF] Neess | Long-chain acyl-CoA esters in metabolism and signaling: Role of acyl-CoA binding proteins[END_REF]. Toxoplasma genome encodes for two ACBPs-cytosolic ACBP1 and mitochondrial ACBP2 [START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF][START_REF] Fu | Synergistic Roles of Acyl-CoA Binding Protein (ACBP1) and Sterol Carrier Protein 2 (SCP2) in Toxoplasma Lipid Metabolism[END_REF]. TgACBP2 binds long chain fatty acids to be transported towards mitochondrial lipid synthesis. This protein is required for the intracellular replication of Type II strain of T. gondii (Prugnaid). In terms of lipid metabolism, the mutant strain PruΔacbp2 has a defect in cardiolipin biosynthesis. The overall abundance of cardiolipin and its potential precursor lipids phosphatidic acid (PA)

and phosphatidyl glycerol (PG), was significantly reduced in the PruΔacbp2 parasites. The expression of an N-terminally HA-tagged MAF1RHb in PruΔacbp2 tachyzoites was able to complement the defect in cardiolipin metabolism [START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF]. The rescue of lipid metabolism defects caused due to loss of TgACBP2, by exogenous MAF1 expression suggests that the host scavenged lipids were able to rescue the loss of de novo biosynthesis.

Toxoplasma has a sterol carrier protein (SCP) called TgHAD-2SCP2, which unlike its other eukaryotic counterparts is expressed in fusion with the enzyme d-3-hydroxyacyl-CoA dehydrogenase and a second SCP-2 [START_REF] Lige | Characterization of a second sterol-esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite[END_REF]. A T.gondii line overexpressing the protein TgHAD-2SCP-2 is able to transport cholesterol and oleate to be incorporated into neutral lipids towards the posterior end of the parasite. Genetic ablation of another FA transporter, TgACBP1 and TgSCP2 together affects parasite replication and attenuates parasite virulence in mice [START_REF] Fu | Synergistic Roles of Acyl-CoA Binding Protein (ACBP1) and Sterol Carrier Protein 2 (SCP2) in Toxoplasma Lipid Metabolism[END_REF]. The experimental demonstration of TgACBP1 dealing mostly with the fatty acids taken up from the host was monitored by uptake of NDB-C16:0. Loss of TgACBP1 from parasites dramatically promoted the uptake of NBD-C16:0-CoA from external environment to compensate for loss of uptake of other FA species from the host. Results obtained by analyzing TgACBP1 mutant using gaschromatography coupled mass spectrometry suggested that there was a specific decline in C18:1 species. Oleic acid is one of the most abundant fatty acid species and it is most likely salvaged from the host (Ramakrishnan et al., 2012;Amiar et al., 2019). TgACBP1 and TgSCP2 collectively provide fatty acyl chains of varying length and saturation for glycerides including triacylglycerols (TAG), diacylglycerol (DAG) and monoacylglycerol (MAG).

e) acquisition of lipids via secretory endocytic pathway

In a study linking T.gondii gliding motility to endocytic-secretory cycle it was shown that lysophosphatidic acid-LPA accumulates in vesicles that are trafficked through the secretory pathway, with a certain accumulation in the VAC, a plant-like parasite organelle and RAB18, a marker of endoplasmic reticulum. This suggests that lipid uptake in these parasites converges with the endocytic secretory pathway. The study also proposed that LPA acts as a physiological stimulator for endocytosis in T. gondii [START_REF] Gras | An endocytic-secretory cycle participates in Toxoplasma gondii in motility[END_REF].

Nolan et al 2017, also found that the host-lipid droplets enriched with neutral lipids endocytosed via the parasite PVM intravacuolar network had Rab7 coating onto their membranes. This also proposes a probable interaction between Rab effectors and proteins present on PVM in order to promote this event of scavenging (Nolan, Romano and Coppens, 2017).

f) Via a molecular sieve between host cell cytoplasm and the parasite, the PVM

The non fusogenic parasite niche that supports intracellular replication of Toxoplasma tachyzoites within its host, called parasitophorous vacoular membrane (PVM) is heavily modified by parasite proteins in order to facilitate import and export of lipidic and polar metabolites. The PVM allows passage of molecules up to 1300-1900 Da between the host cell cytoplasm and the parasite [START_REF] Schwab | The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve[END_REF]. The molecular basis of this porosity is believed to be provided by parasite secreted proteins GRA17 and GRA24 [START_REF] Gold | The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole[END_REF]. (adapted from Fu et al., 2018) and cytoplasmic localizations (TgACBP1, TgSCP1, (adapted from [START_REF] Fu | Synergistic Roles of Acyl-CoA Binding Protein (ACBP1) and Sterol Carrier Protein 2 (SCP2) in Toxoplasma Lipid Metabolism[END_REF] of FA transporters present within the parasite. e) Overview of T. gondii secretory endocytic pathway also assisting in the incorporation of lipids from the external environment (5) (adapted from [START_REF] Gras | An endocytic-secretory cycle participates in Toxoplasma gondii in motility[END_REF]. f) PVM allows passage of small molecules including lipids, with the help of dense granule proteins TgGRA17 and TgGRA23 (adapted from [START_REF] Gold | The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole[END_REF].

The ability of Toxoplasma gondii to infect any nucleated cell resides in the orchestration of its metabolic interaction with the host. These intracellular parasites are in constant competition with their host for nutrient dependencies, which is indirectly dependent on the environmental nutritional conditions [START_REF] Blume | Metabolic interactions between Toxoplasma gondii and its host[END_REF]Amiar et al., 2019).

The host environment, which encompasses lipid metabolites, is one key to unlocking the mystery of species specificity in T. gondii. A recent striking work from Di Genova et al, provides evidence of host metabolic reprogramming allowing parasite co-evolution with its host (Di Genova et al., 2019). This study identified the first metabolic factor responsible for the restricted sexual development of T. gondii solely within feline intestines. Data from this study shows that the accumulation of systemic linoleic acid (18:2) (25%-46% in cat serum) due to the unusual absence of a ∆6 desaturase within feline intestines, allows parasite oocyst propagation. Such a lipid environment therefore provides a unique platform for the development of sexual stages otherwise absent in intermediate hosts. The species barrier was lifted with this key identarian, C18:2 using mice models: wherein by removal of the ∆6 desaturase enzyme from the murine intestine, alongside linoleic acid feeding allowed development of sexual cycle in mice. ∆6 desaturase usually catalyzes the conversion of linoleic acid (C18:2) to arachidonic acid (C20:4) which in turn can participate in formation of immune mediators like eicosanoids. Therefore, absence of this enzyme in feline intestines would also rescue the parasite from of strong immune response from the host.

Another "advantage" of linoleic acid is its ability to perform lipid signaling due to its high susceptibility to oxygenase enzymes thereby generating oxylipins. On the other hand, linoleic acid is toxic for Toxoplasma asexual stage tachyzoites, thus favoring sexual stages in the cat intestine environment.

We recently uncovered that when the parasite is facing adverse host environmental/nutritional conditions, it is capable of sensing the host nutritional status to induce a major metabolic response at two levels : (i) by upregulation of its own de novo lipid synthesis capacities at the level of the apicoplast FASII to boost fatty acid/lipid production, and (ii) concomitant induction of massive changes within the infected host cellular structure by generation of multi-vesicular bodies arising from host organelles via parasite effectors that are then imported to obtain more membrane material (Amiar et al., 2019). In this work, we put T. gondii tachyzoites under nutrient/lipid starvation by growing them under fluctuating levels of fetal bovine serum (FBS) in their culture medium. Lipidomic analysis revealed that the 13 C incorporation into all FASII-generated (C14:0, C16:0) and further ER-elongated FA products (C18:0, C18:1 and C20:1) was significantly increased up to 15% in the absence of FBS in the growth culture medium of the parasites.

Importantly, the FASII which is otherwise known to be dispensable for asexual blood stage P.

falciparum parasites is activated in the presence of lipid deprivation [START_REF] Botté | Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites[END_REF]Amiar et al., 2019). In these conditions the P. falciparum FASII pathway becomes essential during blood stages as confirmed in P. falciparum parasites lacking a functional FASII i.e. FabI knockout.

Together, this data this challenges the current dogma on IPP (isoprenoid precursor) pathway being the sole function of the apicoplast during blood stages. Instead, our findings put the parasite back into its physiological context where nutrient availability and environmental conditions drive the requirement and regulation for a given metabolic pathway.

Toxoplasma tachyzoites, likely for their own benefit, increase the synthesis and overall levels of triacylglycerols (TAGs) in the host thereby resulting in increased host lipid droplet biogenesis (Hu, Binns and Reese, 2017). Parasites can hi-jack the host LD biogenesis via the modulation of transcription of proteins involved in neutral lipid biosynthesis and lipid transport. These included transcriptional upregulation of key enzymes involved in TAG biosynthesis like acylglycerol-3phosphate acyltransferase (AGPAT2), acyl CoA: diacylglycerol acyltransferase (DGAT2), and fatty acid binding transport proteins like FABP3, FABP5. The process is also marked by concordant decrease in the mRNA levels of enzymes involved in TAG lipolysis like ATGL within the host. The parasites require assistance from host mTOR and JNK signaling for the upregulation of LD biogenesis. The HFFs infected with RH∆myr1 parasites and treated with oleic acid showed no change in neutral lipid storage within the host (Hu, Binns and Reese, 2017). MYR1 is a critical parasite secreted protein shown to be involved delivery of effector proteins into the host cells [START_REF] Franco | A novel secreted protein, MYR1, is central to Toxoplasma's manipulation of host cells[END_REF]. Thus, the parasite induced changes in the host neutral lipid metabolism are facilitated by effectors secreted across the PVM into the host as we observed in the case of adaptation to nutrient starvation.

In another interesting study, the authors showed that Toxoplasma replication is reduced in host cells that are depleted of LD, or impaired in TAG lipolysis (Nolan, Romano and Coppens, 2017).

In fact, the host lipid droplets accrued to a perivacuolar localization around the parasite vacuolar membrane until the parasite egresses. Experimental proof of direct uptake of lipids from host LD towards parasites was provided by tracking the trafficking of C4-BODIPY-C9 from host LD to the parasite interior by 24 h post infection. The parasite is are able to manipulate the host endocytic machinery for the uptake of various lipids including diversion of host Rab-labelled vesicles from their Golgi apparatus towards parasite PV lumen for allowing uptake of sphingolipids [START_REF] Romano | Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole[END_REF]. Host Rab7-coated lipid droplets are taken up by the parasite via passage through PV lumen including intravacuolar network (Nolan, Romano and Coppens, 2017).

The mechanism of acquiring the lipids and fatty acids directly from the host can also be induced through host LD-lipophagy. Indeed, Pernas et al. found that Atg-/-murine embryonic fibroblasts (MEF) deficient in macro autophagy were able to accumulate 50% less RC12, a 12-carbon chain saturated FA and BODIPY-FL-C12 (FL C12) [START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. This host autophagy-assisted acquisition of FAs is an essential process for the growth of Toxoplasma within its host.

Toxoplasma proliferation was also reduced in LD-deficient Dgat1-/-,2-/-and LD-lipophagy defective Atg-/-MEFs. The parasite growth defect during its infection in Dgat1-/-,2-/-deficient MEFs was rescued by exogenous addition of cholesterol and fatty acids. This could well mean that the exogenously supplied lipid sources are first metabolized by the host prior to uptake by the parasites as the lipophagy deficient line infected with T. gondii was able to restore the growth defect only partially upon addition of oleate and linoleate.

Toxoplasma type I and type III strains are capable of recruiting host mitochondria in close association with the parasitophorous vacuole, a fascinating process regulated by parasite secreted protein, mitochondria associated factor 1 (MAF1) [START_REF] Pernas | Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response[END_REF]. The parasite MAF1 protein interacts with host cell mitochondrial intermembrane space bridging (MIB) complex [START_REF] Kelly | Toxoplasma gondii MAF1b Binds the Host Cell MIB Complex To Mediate Mitochondrial Association[END_REF]. Later, it was established that the host mitochondrial association was necessary to restrict the intracellular growth of Toxoplasma by limiting its access to fatty acids [START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF].

Toxoplasma type II strains, on the other hand, lack MAF1 and are much less capable of recruiting host mitochondria to their PV. According to this postulate between type I and Type II strains, the knockdown of the mitochondrial acyl-CoA binding protein TgACBP2 was detrimental to type II but did not affect the growth of type I T. gondii strains. This fatty acid transporter protein was

shown to be directly involved in cardiolipin metabolism in type II Prugnaid tachyzoites. Lipidomic analysis of strain lacking ACBP2, PruΔacbp2 complemented with an exogenous copy of MAF1RHb1 showed that MAF1RHb1 expression was able to rescue the reduced abundances of CL and its precursor phospholipids PA and PG [START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF]. This observational study provides a strong link between direct scavenging of lipids from the host organelles, in this case mitochondria, recruited towards the parasite vacuole.

Toxoplasma infection accompanying host organellar remodeling events include the intriguing association of host mitochondria with the parasitophorous vacoular membrane through protein mitochondrial associated factor-1 (MAF-1). Use of Etomoxir, an inhibitor of carnitine-palmitoyl coA transferase and hence β-oxidation within host mitochondria, resulted in better uptake of fatty acids by Toxoplasma [START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. This suggested that the parasites were able to scavenge fatty acids better in the absence of host mitochondrial fatty acid oxidation. By elongation and fusion around the PVM of Toxoplasma vacuole, host mitochondria add another layer of innate immune response by restricting the growth of intracellular parasites via limiting the uptake of FAs.

This embarks a competitive streak between host and the parasite for several metabolites including lipids and fatty acids.

In P. falciparum asexual life cycle the transition from ring to metabolically more active trophozoites boosts major changes in the lipidic composition with an increase in the molar concentration of several phospholipids [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. P. falciparum infected RBCs are able to secrete micro vesicles that have been shown to be involved in intracellular signaling and gametocytogenesis [START_REF] Nantakomol | Circulating red cell-derived microparticles in human malaria[END_REF][START_REF]Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system[END_REF][START_REF] Regev-Rudzki | Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles[END_REF]. These P. falciparum infected RBC-derived micro vesicles (RMVs) were enriched in PS, PI and sphingolipids including ceramide, lactosylceramide (LacCer), dihydroceramide (dhCer) and ganglioside-GM3 [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. It is safe to say that despite being non-proliferative blood stage sexual forms, gametocytes do exhibit high variation in their lipidic relative abundance, suggesting active lipid metabolism. Amongst the lipid species that were increased in the gametocytes included PS, sphingolipids including ceramide. More interestingly the ceramide precursor dihydroceramide was increased by 4-fold in the early and late stages of gametocytes.

Lysophospholipids specifically lysophosphatidylcholine is decreased in the gametocytes. TAGs and TAG stores called lipid droplets were undetectable in the early and late stage gametocytes [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF].

P. falciparum life cycle is dependent on a number of lipid dependent processes including protein trafficking, cell signaling, membrane biogenesis and hemoglobin degradation. The intra erythrocytic blood stage parasite were examined to have at least 2-fold higher levels of phosphatidylglycerol (PG), acyl PG, Lysophosphatidylinositol (LPI), Bis(Monoacylglycerol)Phosphate (BMP), monosialodihexosyl-ganglioside (GM3), diacylglycerol (DAG) and triacylglycerol (TAG) in comparison to uninfected RBC [START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. In the same study the lipids that were enriched in the uninfected RBCs in comparison to the infected ones included phosphatidylserine (PS), phosphatidic acid (PA) and ceramide. These lipids could therefore be serving as a reservoir for the scavenging pathway of the parasite. The sexual stage gametocytes are enriched in sphingomyelin, cholesterol and neutral triglycerides (TAGs).

Amongst the phospholipid species that showed a high increase during gametocyte development included PC 34:0, PC 34:2, and PE36:4 [START_REF] Tran | Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum[END_REF]. Supplementing the growth media of P.

falciparum with polyunsaturated fatty acids is able to induce gametocytogenesis [START_REF] Tanaka | Polyunsaturated fatty acids promote Plasmodium falciparum gametocytogenesis[END_REF]. Another important finding showing the metabolic rewiring during the Plasmodium life cycle is that as the parasite matures, it reduces the incorporation of anapleurotic carbon from glucose and increases incorporation via glutamine [START_REF] Cobbold | Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum[END_REF]. Major findings of this study are summarized below:

i. TgLIPIN is a functional phosphatidic acid phosphatase, essential for Toxoplasma tachyzoite replication and survival within the host.

ii. Parasites lacking TgLIPIN face growth arrest due to severe membrane anomalies mainly affecting inner membrane complex and nuclear envelope.

iii. TgLIPIN, regulates phospholipid biosynthesis feeding parasite membranes by maintaining a balance between its precursor PA and product DAG.

iv.

TgLIPIN metabolizes PA to DAG, which is turn serves as precursor for parasite lipid storage pathway.

v. Most importantly, this key protein channels host scavenged FAs towards membrane biogenesis and storage, preventing their accumulation and thus protecting the parasite from lipotoxicity. * Equal senior and corresponding authors. To whom correspondence should be sent, cyrille.botte@univ-grenoblealpes.fr/cyrille.botte@gmail.com; yoshiki.botte-yamaryo@univ-grenoble-alpes.fr

TITLE: LIPIN, a pivotal nexus in

ABSTRACT

INTRODUCTION

Apicomplexa includes several pathogenic protists that are responsible for major chronic and infectious diseases with massive human and economic burden. Two prominent examples are Toxoplasma gondii and Plasmodium falciparum, the respective causative agents of toxoplasmosis, which affects about 1/3 of the world population, and human malaria, which affects ~200 million people/year killing about 450,000 of the global population (WHO Report 2018). Apicomplexans are obligate intracellular parasites, which have an enormous demand for lipids for sustaining their survival within their human host cells. The utilization of fatty acids (FA) to synthesize the complex lipids is an essential determinant for the successful host adaptation by these parasites. These parasites meet their high demand of lipids through de novo synthesis via type II fatty acid synthesis within apicoplast as well as via copious salvage directly from the host and extracellular environment (Ramakrishnan et al., 2012[START_REF] Ramakrishnan | The intracellular parasite Toxoplasma gondiidepends on the synthesis of longchain and very long-chain unsaturated fatty acids not supplied by the host cell[END_REF]Coppens, 2013;Amiar et al., 2019;Di Genova et al., 2019). Recent data suggests that the tight regulation of FA flux between host, parasite and its pivotal metabolic organelles, potentially through key lipid intermediates, is particularly vital for intracellular development of these apicomplexan pathogens [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]Amiar et al., 2019). For instance, the exposure of Toxoplasma to high concentrations of unsaturated FAs results in irreversible growth arrest due to 'lipotoxicity' [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. However, the molecular and metabolic pathways controlling the FA flux towards parasite membrane biogenesis and storage remain largely unknown.

Phosphatidic acid (PA), the simplest glycerophospholipid, could contribute to the regulation of FA flux in apicomplexans, as it is the key precursor and intermediate balancing the biosynthesis of both glycerophospholipids and triacylglycerols (TAGs) towards membrane biogenesis and lipid storage in eukaryotes (Csaki and Reue, 2010). Apicomplexan parasites can generate PA either (i)

de novo from FA and glycerol-3-phosphate in a two-step acylation reaction catalyzed by glycerol-3-phosphate acyltransferases (GPAT/ATS1) and acylglycerol acyltransferases (AGPAT/LPAAT/ATS2), respectively [START_REF] Lindner | Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development[END_REF]Amiar et al., 2016Amiar et al., , 2019;;Shears et al., 2017), and (ii) from existing glycerolipids by diacylglycerol kinases (DGK1 and DGK2) or phospholipase D (Bullen et al., 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF]. PA has pleiotropic roles depending on its biosynthetic source and the site of production within the parasite, (i) as a signal transducer modulating parasite invasion, motility and egress (Bullen et al., 2016;Jacot et al., 2016;Jimah et al., 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF], (ii) as a regulator of lysoPA (LPA)/PA levels for modulating membrane curvature for cytokinesis and endocytosis (Amiar et al., 2019), and (iii) as the central precursor of phospholipid synthesis in the apicoplast/ER pathway formed by ATS1 and ATS2 (Amiar et al., 2016(Amiar et al., , 2019)).

Since, PA appears at the forefront of parasite biological armor, therefore, the enzymes involved in its metabolism are critical for our understanding of parasite pathogenesis. PA can be catabolized to another key signaling lipid diacylglycerol (DAG), by phosphatidic acid phosphatases (PAP), which play key metabolic functions in eukaryotes (Carman and Han, 2019). Apicomplexan parasites possess three putative PAPs although none of them has been characterized to date.

Here, we determined that Toxoplasma lipin, TgLIPIN, is a phosphatidic acid phosphatase that localizes at the cytosolic-ER interface and is essential for the intracellular survival of tachyzoites.

Its inducible disruption immediately leads to gross membrane anomalies at the parasite IMC and nucleus. Lipidomics of the inducible knockdown mutant reveals a time-dependent accumulation of PA concomitant with the reduction of DAG, lipid droplets and storage lipid triacylglycerol (TAG). Consequently, the mutant dies from a lipotoxic accumulation of free FA and membrane phospholipids. Further, we conducted novel fluxomics experiments using U-13 C glucose-labelled host cells to monitor which FA are being salvaged by the parasite, providing the first host scavenged lipidome of Toxoplasma. This allowed to determine that TgLIPIN acts as the central metabolic point that tightly controls the flux of host-derived FA for the synthesis and regulation of phospholipids vs TAG towards membrane biogenesis and storage, necessary for intracellular parasite development. We therefore unravel the molecular mechanism by which the parasite uses both host and de novo synthesized lipid resources to generate the lipids required for its survival.

RESULTS

Toxoplasma genome encodes a single lipin, TgLIPIN, which has functional phosphatidate phosphatase activity

Bioinformatics analysis of TgLIPIN (TGGT1_230690) revealed that the protein has an unusually large size compared to other phosphatidate phosphatases encoded by the genome of T. gondii.

TgLIPIN possesses the two typical and highly conserved domains of eukaryotic lipins, the amino terminal N-LIP domain, and the carboxy terminal C-LIP domain harboring its functional PA phosphatase catalytic motif DXDXT/V (HAD-like domain) (Fig. 1a) [START_REF] Carman | Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis[END_REF].

Phylogenetic analysis confirms that the enzyme is highly conserved as a single lipin within phylum apicomplexa, cladding specifically within a coccidian subgroup. (Sup Fig. 1a).

To confirm the predicted phosphatidic acid phosphatase activity of TgLIPIN, we performed heterologous complementation of a Saccharomyces cerevisiae ∆dpp1∆lpp1∆pah1 triple mutant deficient in PAP activity with a temperature sensitive phenotype not allowing growth at 37ºC [START_REF] Nakamura | Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation[END_REF][START_REF] Chae | The Saccharomyces cerevisiae actin patch protein app1p is a phosphatidate phosphatase enzyme[END_REF]. The C-LIP domain of TgLIPIN was able to rescue the temperature sensitive phenotype in the yeast mutant (Fig. 1b) thereby confirming presence of PAP enzymatic activity.

Next, TgLIPIN was endogenously tagged with 3×HA at its C-terminal end (Huynh and Carruthers, 2009). Immunofluorescence assay (IFA) revealed a broad cytosolic and perinuclear localization.

(Fig. 1c). Interestingly TgLIPIN lacks an apparent nuclear localization signal (NLS) that most of eukaryotic lipin usually possess (Csaki and Reue, 2010;Zhang and Reue, 2017). To further resolve its localization, we conducted IFAs of TgLIPIN-HA with a known Toxoplasma ER marker, Der1-GFP [START_REF] Agrawal | Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins[END_REF], which confirmed proximity to the endomembrane system with partial ER co-localization (Fig. S1b,c). We then sought to assess the localization of the putative P. falciparum lipin, PfLIPIN via endogenous tagging. Unlike TgLIPIN, the Plasmodium lipin is predicted to have a nuclear localization (NLS) sequence. IFAs confirmed that PfLIPIN was specifically expressed during the schizont stage of the parasite erythrocytic life cycle, as punctate vesicles in close vicinity to, or at the parasite nucleus (Fig. S3d).

TgLIPIN disruption induces rapid division defects leading to replication arrest and parasite death

To understand the importance of TgLIPIN for parasite growth, we generated an N-terminal HAtagged inducible knockdown parasite line based on Tet-off system, TgLIPIN-iKD (Meissner, Schlüter and Soldati, 2002;Sheiner et al., 2011) (Fig. S2a,b). Downregulation of TgLIPIN-iKD showed no detectable protein by western blot after 48h of anhydrotetracycline (ATc) treatment (Fig. 2a). Cytoplasmic localization of the protein and its downregulation were both confirmed by IFA, which also revealed gross parasite malformation after 48h of ATc treatment (Fig. 2b). This result suggests that TgLIPIN disruption dramatically impacts parasite development within its host.

To further delineate the morphological phenotype of TgLIPIN downregulation, IFAs were performed prior to complete protein loss, at 24 h of ATc treatment (Fig. 2c). Severe membrane anomalies and division defects were observed at this early time point of ATc treatment, suggesting

TgLIPIN levels are crucial to maintain parasite intracellular growth. Co-localization with known parasite inner membrane complex marker GAP45, suggested a gross defect in IMC biogenesis (Fig. 2c, enlarged image). This irregular membrane biogenesis caused a severe intracellular replication defect in TgLIPIN-ikD with a significant increase of small vacuoles containing 1-2 parasite vacuoles (15-20%) and vacuoles containing morphologically abnormal parasites (10-15%), alongside with a concomitant significant decrease (25-80%) of larger vacuoles containing 3-10 parasites (Fig. 2d). To further assess the effect of TgLIPIN on parasite intracellular growth, plaque assays were performed investigating the mutant capacity to maintain proper growth with fluctuating levels of host nutrients. When cultured in regular growth conditions with 1% FBS, TgLIPIN-ikD (+ATc) exhibited a severe growth defect with few plaques (Fig. 2e) as expected from IFA results (Fig. 2b,c). In an attempt to rescue this defect, the parasites were grown in the presence of 10% FBS (Amiar et al., 2019). However, surprisingly, TgLIPIN-ikD (+ATc) showed a significant decline in growth in 10% than with 1% FBS containing culture medium, as marked by complete absence of plaques (Fig. 2e,f). In contrast, a decrease in host nutritional environment with 0% FBS slightly and significantly enhanced the growth of TgLIPIN-ikD (+ATc) (Fig. 2e,f)

suggesting that the exogenous lipidic nutrient source such as free FA or phospholipids are somehow toxic to the parasites lacking TgLIPIN. This data is an example of robust adaptation of parasite to its host nutritional environment.

Electron microscopy reveals gross membrane anomalies as an early impact of TgLIPIN downregulation

In order to further resolve the cellular phenotype marked by the TgLIPIN down regulation,

TgLIPIN-iKD (+ATc) parasites were examined by transmission electron microscopy (TEM).

Interestingly, as early as 24 h treatment with ATc , when TgLIPIN levels are only slightly reduced, both parasite inner membrane complex (IMC) and plasma membrane displayed gross abnormalities in forming evaginations or lateral interruptions, without affecting the parasite size ( Fig. 3 b ). Concordantly, IFA data of TgLIPIN-ikD (+ATc) also hinted at aberrant IMC (Fig. 3c). The nuclear envelope membranes also imbibed a grave effect early in the process of TgLIPIN down regulation (24 h +ATc). The nuclear membrane appeared multi-lobed marked by abnormal extensions into the cytoplasm (Fig. 3d). Interestingly, this nuclear envelope phenotype is also observed in the yeast lipin mutant coupling phospholipid biosynthesis to nuclear membrane [START_REF] Santos-Rosa | The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth[END_REF][START_REF] Siniossoglou | Lipins, lipids and nuclear envelope structure[END_REF].

Furthermore, observations of TgLIPIN-iKD at 24 h +ATc showed daughter cells abnormally attached through IMC (Fig. 3c, right). In agreement with the replication assay data (Fig. 2d), TEM images showed that the process of parasite endodyogeny was affected by the absence of appropriate levels of TgLIPIN (24 h +ATc), marked by arrest of multiple daughter cells within a single mother cell (Fig. 3e). This suggests that indispensability of TgLIPIN for parasite growth is based on its direct involvement in endomembrane biogenesis (i.e. plasma membrane, IMC, nucleus), in turn affecting the process of parasite replication within host cell.

TgLIPIN regulates the synthesis of glycerophospholipids by controlling the bulk synthesis of PA and DAG

To determine the functional role of TgLIPIN in parasite PA synthesis and membrane biogenesis, we conducted comprehensive lipidomic analysis. To precisely assess the timely impact of TgLIPIN-ikD disruption on lipid synthesis, parasites were grown for 24 to 48 h with and without ATc. Total lipids were separated by high performance thin layer chromatography (HPTLC) and quantified by gas chromatography-mass spectrometry (GC-MS).

PA levels were significantly increased to almost three times early on at 24 h + ATc in TgLIPIN-ikD in comparison to the control (-ATc) (Fig. 4b). Such an increase was maintained after 48 h of TgLIPIN downregulation (Fig. 4b). Analysis of the FA composition of PA molecular species revealed that the disruption of TgLIPIN showed a significant increase in the level of unsaturated fatty acid oleate (C18:1) besides decline in levels of various saturated fatty acids, significantly stearate (C18:0) (Fig. 4c). The amount of DAG, the product of TgLIPIN, was significantly reduced to almost half in the TgLIPIN-ikD (+ATc), beginning from 24 h up to 48 h treatment with ATc (Fig. 4d). The concomitant increase in PA and decrease in DAG indicated that TgLIPIN is an active phosphatidic acid phosphatase, controlling the bulk levels of PA and DAG. Despite a strong replication defect and reduction in parasite number, the total amount of phospholipids was increased to almost twice in the TgLIPIN-ikD (+ATc) in comparison to the control (-ATc) (Fig. 4e). Accordingly, the phospholipid classes directly made from PA via the CDP-DAG pathway (Kong et al., 2017) were found significantly increased, i.e. cardiolipin (CL) and phosphatidylserine (PS) and indirectly phosphatidylcholine (PC) likely formed from PS and phosphatidylethanolamine (PE) (Fig. S4a).

TgLIPIN controls critical levels of DAG and free fatty acids towards parasite storage lipids, triacylglycerols

In eukaryotes TAG biosynthesis occurs at the last step of the glycerol-3-phosphate pathway through the sn-3 acylation of DAG catalysed by a diacylglycerol-acyltransferase (DGAT) that uses FFA [START_REF] Cases | Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis[END_REF]Nolan, Romano and Coppens, 2017;[START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. However, the key and limiting step of TAG synthesis is the formation of its precursor, DAG, the production of which in turn is dependent on a phosphatidate phosphatase (lipin). We tested the role of TgLIPIN in this key process by comparing the levels of neutral lipid majorly comprising TAGs and cholesteryl esters within reserve organelles called lipid droplets by nile red staining [START_REF] Greenspan | Nile red: A selective fluorescent stain for intracellular lipid droplets[END_REF] in the TgLIPIN ikD parasites (+ATc/-ATc). Nile red staining showed that the number of lipid droplets per parasite vacuole in the presence of ATc was reduced to almost 60% in comparison to the control (Fig. 5a,b), suggesting that TgLIPIN is involved in the synthesis of storage lipids. Accordingly, relative abundance of TAGs was significantly reduced after 48 h of ATc treatment, with a 24 h time lag compared to DAG levels (Fig. 5c). Other neutral lipids making the bulk of lipid droplets, cholesteryl esters (CE), were also significantly decreased (Fig. S4c).

TAGs and CEs are neutral storage lipids potentially implied in the parasite's ability to cope with the excess FA [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. The synthesis of TAG via enzyme DGAT requires two substrates, DAG and free fatty acids (FFA). We therefore determined the FFA content of the TgLIPIN-ikD. Lipidomics revealed a significant accumulation of FFA in the TgLIPIN-iKD (+ATc/-ATc), increasing with time points 24 to 48 h (Fig. 5d). Interestingly, similar to PA, C18:1 (oleate) was the only FA found significantly increased in the parasite free FA (FFA) pool (Fig. S3), suggesting this FA is an important intermediate in the pathways regulated by TgLIPIN, as previously hinted (Hu, Binns and Reese, 2017;[START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF][START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF].

Therefore, this key result suggests cell death in parasites lacking TgLIPIN is caused due to 'lipotoxicity' or toxic effect of FFAs, arising because of impaired TAG biosynthesis. This is consistent with the reduced viability of parasite in high FBS content (Fig. 2e,f).

TgLIPIN regulates the flux of host fatty acids to control parasite lipid synthesis and prevent accumulation of toxic free fatty acids levels

Previous results from this study suggest that TgLIPIN is involved in DAG synthesis and regulation of PA levels, further channeling fatty acids towards TAG and/or phospholipid biosynthesis. Fatty acids required for lipid synthesis in T.gondii are either obtained from the host (Ramakrishnan et al., 2012(Ramakrishnan et al., , 2015;;Nolan, Romano and Coppens, 2017;[START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]Amiar et al., 2019) and/or synthesized de novo via the apicoplast FASII (Mazumdar and Striepen, 2007;Amiar et al., 2016).

In order to identify the source of increased phospholipids and toxic FA observed upon TgLIPIN depletion, we set up fluxomics approaches where we grew parasites and/or host cells using different stable isotope substrates-containing media ( 13 C-U-Glucose, 31 d-C16:0) and performed lipidomic analyses for each experimental setup (Fig. 6a,b,c) .

First, parasites were labelled with 13 C-U-Glucose, which was added to a glucose free medium, to confluent host cells together with parasites with or without ATc to monitor de novo FASII activity (Fig. 6a). Total lipid content, including both FFA and FA originating from glycerolipids, was extracted to determine the ratio of 13 C incorporation to each FA as a measure of FASII activity. In TgLIPIN-ikD (+ATc), the amount of labelled FA made by FASII was overall similar to the control (-ATc) in terms of both total lipid content (Fig. 6d) and the FFA pool (Fig. 6e). This suggested that both phospholipid and FFA increase in TgLIPIN deficient strain does not rely on the de novo fatty acid synthesis. We then assessed the FASII activity in both normal and TgLIPIN depleted parasites. In the TgLIPIN-ikD (+ATc), there was a significant reduction in percentage of 13 C labelling in C14:0, the major FASII product (Fig. 6f). Distribution of 13 C incorporation to each isotopologue of FASII FA product confirmed that the pathway was nevertheless functional (Fig S5a,b). This suggests that although the TgLIPIN depleted parasites do not source FA from FASII, its activity is slightly reduced in the mutant.

In order to monitor the FAs sourced from the external environment/culture medium, we grew parasites and confluent host cells in a medium supplemented with deuterated palmitic acid ( 31 d-C16:0) (Fig. 6c. Parasites were harvested and total lipids were extracted to determine the incorporation of 31 d-C16:0 in parasite lipids. In the total lipid content, 31 d-C16:0 and its elongation/desaturation product 29 d-C18:1 were detected in a similar amount in the TgLIPIN-ikD parasites with or without ATc (Fig. 6g). However, a reduction in the d31-C16:0 incorporation to FFA species within TgLIPIN-ikD (+ATc) in comparison to the control (-ATc), further abolished host external environment as the source of increased lipids within the mutant (Fig. 6h).

To monitor the fatty acids directly scavenged from the host, we designed a novel assay using U-13 C-Glc to label host lipids (Fig. 6b). Non confluent host cells were grown in the presence of medium containing U- 13 C-Glc which would theoretically fuel any of the active FA synthesis pathways within the host (FASI, elongases) via the synthesis of their substrates, acetyl-CoA, thereby generating 13 C-pre-labelled host metabolites including lipids and fatty acids [START_REF] Mashima | De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy[END_REF]. These 13 C-pre-labelled host cells were then infected with parasites in the presence of normal culture medium, which contains regular 12 C-Glc. Then total lipid was extracted from the parasites to determine the ratio of 13 C incorporation to each fatty acids to determine the origin from the host or not. Using this novel approach, we were able to determine (i) the FA biosynthetic capacities of the host cells (Fig. 6i), and more importantly (ii) the first scavenged FA lipidome of the parasite (Fig. 6j). Host, human foreskin fibroblast (HFF), are capable of synthesizing FA ranging from C14:0 to C20:1, with the most abundant FA species being C18:1 (Fig. 6i). The wild type (TgLIPIN-ikD -ATc) parasites are capable of scavenging all these FA species made by the host cell with a major preference again for C18:1 (Fig. 6j). Importantly, in TgLIPIN-ikD +ATc parasites, both total lipid and FFA exhibited significantly increased 13 C incorporation from host FA in comparison to the control strain (-ATc) (Fig. 6j). This strongly supports that the origin of FA source for the excess phospholipids and FFA in TgLIPIN-iKD+ATc is the host (Fig. 4e,5d). In terms of individual FA species, C16:0 and C18:0 were significantly increased in the FFAs derived from the TgLIPIN mutant (+ATc) (Fig 6k). Furthermore, analysis of the molecular species in the most abundant phospholipid, PC, showed a significant increase of host derived C18:1 in TgLIPIN mutant (+ATc) (Fig 6l). Together, this data correlating with our previous results also suggests that the TgLIPIN specifically fuels PA enriched in oleic acid (C18:1) towards phospholipid synthesis and simultaneously channels DAG and FFAs-C16:0/18:0 for TAG synthesis.

DISCUSSION

Our data here shows that TgLIPIN is a phosphatidate phosphatase controlling the flux of FAs towards either phospholipid synthesis or storage, disruption of which has multiple phenotypic consequences within the parasite (Fig. 7).

In Saccharomyces cerevisiae, lipin acts as a Mg2+ dependent phosphatidate phosphatase catalyzing the biochemical conversion of PA to DAG in the route of glycerol-3-phosphate [START_REF] Carman | Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis[END_REF]. In mammalian cells, lipin is encoded by three independent genes lipin1, lipin2 and lipin3, all of which have PA phosphatase activity modulating the levels of TAGs and phospholipids (Zhang and Reue, 2017). Genetic mutation within these lipins causes severe metabolic disorders including rhabdomyolysis (lipin1), obesity (lipin1), autoinflammatory disease (lipin2) and impaired lipoprotein assembly in the intestines (lipin2 and 3) (Reue and Wang, 2019).

The essentiality of lipin for T. gondii growth also suggests distinct function from the two other parasite PAPs. Neither Toxoplasma PAPs (TGGT1_247360 and TGGT1_246490) appeared to compensate for the loss of TgLIPIN, which is in contrast to yeast in which deletion of gene encoding lipin was compensated, at least in part, by other PA phosphatases [START_REF] Chae | The Saccharomyces cerevisiae actin patch protein app1p is a phosphatidate phosphatase enzyme[END_REF].TgLIPIN was localized widely to parasite cytosol overlapping endomembrane compartments. In other eukaryotic systems, the cytosolic lipin proteins are able to translocate to various membrane depending on the availability of their substrate PA and/or in response to stimuli such as fatty acids (Zhang and Reue, 2017). These phosphatidate phosphatases also translocate to the nuclear compartment and act as transcriptional regulators, e.g. lipin1 acts as an inducible amplifier of PGC1 alpha/PPAR alpha pathway in hepatocytes (Finck et al., 2006). Like its human counterpart, yeast Pah1 has implications in UASINO-containing genes, and the growth of the nuclear/ER membrane [START_REF] Carman | Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis[END_REF]. Therefore, it is conceivable that TgLIPIN might be quite dynamic and the perinuclear localization we see in part might be more prominent if it is activated more so to embed it into the ER, nuclear membrane. The TgLIPIN mutant hints at an probable link between metabolism and the nucleus of the parasite.

Metabolic regulation of TgLIPIN

Of note, unlike other enzymes of the TAG biosynthetic pathway, Pah1 and lipins lack transmembrane domains and therefore must first translocate onto membranes in order to generate DAG. This step is inhibited by multisite phosphorylation, mediated by Pho85, Cdc28, and protein kinase A in yeast [START_REF] Choi | Pho85p-Pho80p phosphorylation of yeast pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism[END_REF][START_REF] Su | Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-Cyclin B kinases to regulate lipid synthesis in yeast[END_REF] and target of rapamycin (TOR) and mitotic kinases in mammals [START_REF] Harris | Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1[END_REF][START_REF] Grimsey | Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2[END_REF]. In continuation with this, we observed a hypophosphorylation at a serine residue (amino acid position-1019) of TgLIPIN protein in a phospho-proteomic screen of a cyclin-dependent protein kinase-TgCDPK7 knockout strain (unpublished data). This suggests the possibility of TgCDPK7 as the kinase regulating TgLIPIN via phosphorylation.

In contrast, dephosphorylation of Pah1 in yeast is catalysed by the highly conserved transmembrane Nem1-Spo7 complex [START_REF] Siniossoglou | A novel complex of membrane proteins required for formation of a spherical nucleus[END_REF][START_REF] Santos-Rosa | The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth[END_REF], which is also required for Pah1 membrane recruitment and activation [START_REF] O'hara | Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase[END_REF][START_REF] Karanasios | A phosphorylation-regulated amphipathic helix controls the membrane translocation and function of the yeast phosphatidate phosphatase[END_REF][START_REF] Choi | Pho85p-Pho80p phosphorylation of yeast pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism[END_REF]. Dullard, a homolog of Nem1p in mammalian cells is involved in dephosphorylation of phosphatidate phosphatase, which further generates a phosphate cascade consequently regulating nuclear membrane biogenesis [START_REF] Kim | A conserved phosphatase cascade that regulates nuclear membrane biogenesis[END_REF]. Using bioinformatic screen, we identified the T. gondii Nem1p/Dullard homolog and named it TgDULL. TgDULL is a predicted protein phosphatase harboring a HAD domain and upon endogenous tagging with 3xHA, the protein localized to the endoplasmic reticulum of the parasite (Fig. S7). The protein functional domain and localization of TgDULL present it as a strong candidate as the master regulator of TgLIPIN. This however needs further experimental proof showing an effect on the phosphorylation status and localization of TgLIPIN upon genetic disruption of TgDULL.

Implications of lipid changes on phenotype of TgLIPIN mutant: Phospholipid and TAG biosynthesis

One of the early phenotypic stress evoked in TgLIPIN mutant was its impact on major membranes including the nuclear envelope complexed with the ER and the parasite inner membrane complex.

Downregulation of lipin and its homologs in other eukaryotes impacts nuclear membrane biogenesis. In addition to their critical role in yeast lipid metabolism, mutant strains lacking pah1 gene display a striking expansion of the ER and the nuclear membrane. [START_REF] Santos-Rosa | The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth[END_REF][START_REF] Siniossoglou | Lipins, lipids and nuclear envelope structure[END_REF]. Fission yeast cells lacking the Pah1 orthologue Ned1 also display the peculiar nuclear defect [START_REF] Tange | An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin[END_REF]. There are evidences that suggest that the nuclear membrane biogenesis is directly linked to PA metabolism. In ∆pah1 yeast, mutations in upstream biosynthetic steps of the glycerolipid pathway that lower PA levels also reduced the aberrant expansion of nuclear membrane [START_REF] Han | An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth[END_REF]. Effects of pah1 deletion could be phenocopied by overexpression of Dgk1p, a novel nuclear/ER membrane DAG kinase that generates PA [START_REF] Han | An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth[END_REF]. Catalytically inactive Pah1 retains its nuclear membrane capability, but lipid precursors are redirected toward phospholipids instead of storage TAGs, thereby resulting in deformation of nuclear membrane [START_REF] Barbosa | Lipid partitioning at the nuclear envelope controls membrane biogenesis[END_REF]. Another hypothesis for the same could be increased phospholipid biosynthesis due to PA accumulation via the CDP-DAG pathway (Fig. S4a), as has also been shown in ∆pah1 yeast [START_REF] Han | An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth[END_REF].

In apicomplexa biology, a recent study demonstrated that use of DGAT inhibitor-T863 to inhibit lipid storage capacity in T. gondii resulted in rapid accumulation of membranous structure accompanied by rapid parasite death due to strong replication defect [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. This cytopathy was restricted mainly to the endomembrane system, i.e. ER and the associated nuclear envelope, during the early time points of treatment, similarly as in case of TgLIPIN depletion. The electron micrographs of the TgLIPIN mutant (+ATc) clearly suggest that the replication arrest was due to accumulation of excessive membranes within the parasite. The replication arrest within the parasite is possibly directly linked to aberrant IMC biogenesis. The IMC is one of the first membranes recycled directly from the mother cell into progeny, thus playing key role in the process of parasite endodyogeny (Ouologuem and Roos, 2014).

Phosphatidic acid is the central precursor for all glycerophospholipids as well as triglycerides thereby residing at a pivotal metabolic bifurcation point. In the TgLIPIN mutant an accumulation of PA redirects the pathway towards increase in phospholipids via the liponucleotide cytidine diphosphate-diacylglycerol (CDP-DAG). This is complemented by the utilization of excess free fatty acids into phospholipids, partially circumventing their inability to be sequestered into storage triglycerides. We observed an overall increase in the major membrane phospholipids including cardiolipin (CL) and phosphatidylserine (PS) which are indeed derivatives of the CDP-DAG pathway in other eukaryotes [START_REF] Fakas | DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae[END_REF]. Other than this we also observed an increase in the major membrane phospholipid PC, which could be channeled via increased PS through Kennedy pathway. Based on existing literature another plausible explanation of lipids excess could be the involvement of increased PA levels in de-repression of phospholipid biosynthesis genes expression, as has been reported in eukaryotic model yeast. The yeast lipin mutant pah1Δ is characterized by accumulation of PA which contributes to increased phospholipid biosynthesis by sequestering transcriptional oppressor Opi1acting against transcriptional activators Ino2p/Ino4p [START_REF] Han | The Saccharomyces cerevisiae lipin homolog is a Mg2+dependent phosphatidate phosphatase enzyme[END_REF][START_REF] Han | The cellular functions of the yeast lipin homolog Pah1p are dependent on its phosphatidate phosphatase activity[END_REF].

Additionally, Pah1 has also been reported to be a part of the protein complex that binds DNA to block transcription from promoters of genes transcriptionally controlled by INO1 and OPI1 (Santos-Rosa et al., 2005). In Toxoplasma, an immunoprecipitation with the HA-tagged TgLIPIN could provide major insights into its possible role in transcriptional regulation of genes involved in phospholipid biosynthesis. By defining the protein-interacting partners of TgLIPIN, several regulatory mechanisms can also be elucidated.

The triacylglycerol content in TgLIPIN mutant decreases only 48 h post protein downregulation with ATc suggesting that due to decrease in the substrate-specific DAG species because of TgLIPIN unavailability, the parasite is unable to acylate the free fatty acids to generate its subsequent product TAG. We did not detect any significant changes between the cholesteryl ester species (Fig. S4c) in the mutant vs wild type strain, further strengthening the reaction specificity of TgLIPIN towards TAG biosynthesis.

In mammalian cells the inhibition of triglyceride synthesis leads to oleic acid (C18:1) induced lipotoxicity [START_REF] Listenberger | Triglyceride accumulation protects against fatty acid-induced lipotoxicity[END_REF]. Thus, lipid droplets act as more than just cellular energy reserves by protecting the cells from deleterious effects of toxic excess free fatty acids by their incorporation into TAGs.

Nutrient sensing and host mediated remodeling of parasite lipids

By pre-labelling the host fibroblast cells with U-13 C-Glucose, we were able to track the source of excess fatty acid and phospholipid content in the TgLIPIN mutant. In keeping with previously reported data, using 13 C-fluxomics we observed that the parasite has a specific preference for scavenging oleic acid (C18:1) directly from host [START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. Existing literature provides a strong evidence of involvement of C18:1 is TAG biosynthesis and subsequent LD formation in mammals [START_REF] Listenberger | Triglyceride accumulation protects against fatty acid-induced lipotoxicity[END_REF] as well as parasites [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. Thus, the excess oleate within the parasite sourced directly from the host has a strong correlation to the lipid storage defect in TgLIPIN mutant. In T. gondii the salvage of C18:1 from host environment is in part utilized for phospholipid synthesis resulting in extra membranous structures, further impeding the process of endodyogeny [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. With the model of TgLIPIN parasite mutant we propose that the host participates in active remodeling of parasite phospholipids as well as neutral lipids. This is directly rooted to our observation of the difference between phosphatidic acid FA molecular species in the mutant in comparison to the wild type, with a significant rise in the levels of C18:1 specifically in the mutant. Such phospholipid remodeling has also been reported in the yeast mutant where deletion of Pah1 results in an increase (22-27%) in palmitoleic acid and a decrease (17-21%) in oleic acid [START_REF] Fakas | DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae[END_REF]. We were able to detect an increase in the host-derived ( 13 C labelled) oleic acid within the parasite phosphatidylcholine.

However, we could not detect any 13 C labelling in the parasite PA. The plausible explanation behind this is the dynamic nature of PA that is rapidly utilized for membrane phospholipids and other signaling derived events.

Meanwhile, the reduction in prokaryotic type-FASII activity in response to increased amount of fatty acids within the parasite suggests adaptation of parasite to nutrient sensing. In the case of TgLIPIN mutant an increase of fatty acids derived from host signals the parasite to stagnate its FASII activity. Similar observations on metabolic plasticity show that there is a reduction in growth of P. falciparum FASII KO mutant in the presence of nutrient-deprived culture conditions (Amiar et al., 2019). Another study in bacteria shows that the FASII pathway is repressed in a negative feedback loop mediated by acyl-CoA [START_REF] Fujita | Regulation of fatty acid metabolism in bacteria[END_REF]. Taken together, it can be concluded that TgLIPIN is one of key proteins regulating major fatty acid fluxes from the host as well as apicoplast into parasite membrane and storage lipids. Furthermore, indispensability of TgLIPIN suggests the importance of maintaining the lipid homeostasis by control of critical levels of PA.

METHODS AND MATERIALS

Sequence analysis and structure generation

TgLIPIN (TGGT1_230690) and PfLIPIN (Pf3D7_0302300) were identified using EuPathDB web sources ToxoDB (http://toxodb.org/toxo/) and PlasmoDB (http://plasmodb.org/plasmo/) respectively. A phylogenetic tree of lipin proteins in several eukaryotes was created using the online platform Phylogeny.fr. The organisms used for lipin protein sequences for generation of the phylogenetic tree include: Toxoplasma gondii (TGGT1_230690), Plasmodium falciparum

T. gondii strains and cultures

The parasite host cells human foreskin fibroblasts (HFF) were cultured using Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and 25 μg/mL gentamicin (Gibco) at 37°C and 5% CO2.

T.gondii tachyzoite parental strains RH-ΔKu80 TATi, RH-ΔKu80 as well as mutant strains

TgLIPIN-iKD, TgLIPIN-3*HA were propagated by serial passage within their host HFF using DMEM supplemented with 1% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and 25 μg/mL gentamicin (Gibco) at 37°C and 5% CO2.

Generation of HA-tagged and inducible knockdown line for TgLIPIN

A C-terminally tagged HA line was generated expressing from the gene's endogenous locus using the classical pLIC strategy using homologous recombination in a RH-ΔKu80 strain. For the same, a 1677 bp homology region of TgLIPIN located towards to C-terminus excluding the stop codon, was amplified from the parasite genomic DNA using the primers forward 5'-TACTTCCAATCCAATTTAATGCACGGCAGATTTCTCTTACTGG-3' and reverse 5'-TCCTCCACTTCCAATTTTAGCCAAATTACTGCATTTGCGTTCAC-3'. The homology region was assembled into PacI digested pLIC-HA-DHFR plasmid using ligation independent cloning protocol (Huynh and Carruthers, 2009). The assembled plasmid with linearized with single enzyme site specific to the parasite DNA sequence within the plasmid-NsiI just before transfection.

Parasites were selected with the drug pyrimethamine and cloned by limiting dilution.

For generation of inducible knockdown plasmid pPR2-DHFR (Katris et al., 2014)

Generation of HA-tagged and inducible knockdown line for PfLIPIN

A transgenic P. falciparum line was generated using riboswitch based glmS system in which et al., 2017). Asexual ring stage 3D7 parasites were transfected with the final construct PfLIPINptex-HAglmS. Transgenic parasites were selected with 2.5 nM WR99210 (Jacobus).

The positive parasite population was screened using the primers, P1 5'-GATAGAATCCGAGTAAGATATCG-3', P2 5'-CATCGTACGGATACGCATAAT-3', P3 5'-CTTGGTTTGAAGAAATCCTTACG-3', P4 5'-CGAACATTAAGCTGCCATATC-3', P5 5'-GATGCAGTTTAGCGAACCA-3' in the combinations described in Fig S2 . All PCRs were performed using TaKara primestar GXL polymerase.

Immunofluorescence assay

Primary antibodies anti-HA (Rat, Roche) , anti-IMC1 (Mouse), anti-GAP45 (Mouse) were used at dilutions 1:500, 1:1000 and 1:1000 respectively. Secondary AlexaFluor 488-and 546-conjugated anti-mouse, anti-rat and anti-rabbit antibodies (Life Technologies) were used at 1/2500. For the immunofluorescence assay (IFA) parasites were grown on confluent HFF on coverslips and fixed in PBS containing 2.5% paraformaldehyde (PFA) for 15 min at room temperature (RT). Samples were permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT prior to blocking in PBS containing 3% BSA and subsequent incubation with primary antibodies then secondary antibodies diluted in the blocking solution. Labelled parasites were stained with Hoechst (1/10000, Life technologies) for 20 min and then washed three times in PBS before final mounting of the coverslips on a glass slide using fluorogel. The IFA slides were visualized using fluorescence microscope (Axio Imager 2_apotome; ZEISS).

Western blot analysis

Parasites were harvested for western blot after complete egress from their host. In order to remove any host cell debris, the parasites were passed through a 3 μm filter, then counted by hemocytometer and solubilized in SDS buffer at equivalent cell densities. Equal amount of protein was separated on a 4-12% gradient SDS-polyacrylamide (Life Technologies) and transferred to Nitrocellulose membrane (check this) using the XCellII Blot Module (Invitrogen). Primary antibodies anti-HA (Rat, Roche) and anti-TOM40 (Rabbit, Geil G Van Dooren et al 2016 JBC) were used at a dilution of 1:500 and 1:1000, respectively. Secondary goat anti-mouse and antirabbit horse radish peroxidase (HRP) conjugated antibodies (Thermo Scientific) were used at 1:2000. Protein signal was detected by chemiluminescence after membrane staining with luminata crescendo western HRP detection kit (Millipore). The signal strength of protein was quantified using a BioRad chemidoc imager (BioRad).

Phenotypic analysis

Plaque assay-The extracellular parasites were harvested after filtration and counted by hemocytometer. Then approx. 500 parasites were inoculated to confluent HFF flask (25 cm 2 ).

TgACS3-ikD was grown for plaque assay in the presence or absence of ATc (0.5 μg ml -1 ) for 7-10 days. Plaque sizes were visualized by crystal violet staining (30-60 min) after aspiration of culture media, and cells fixation with 100% ethanol (5 min) followed by phosphate-buffered saline (PBS) wash.

Replication assay-The parasites were grown for two days with or without ATc (0.5 μg ml -1 ), harvested and filtered. Equal number of parasites were allowed to invade confluent HFF grown on coverslips. Following 2 h of invasion, the coverslips were washed thrice with ED1 (1% FBS containing DMEM), in order to remove any uninvaded parasites and promote synchronized replication. Anhydrotetracycline (ATc) (0.5 μg ml -1 ) was added at the outset of the experiment, allowing the treatment for 24 h, alongside control parasites without ATc. These coverslips were then fixed and processed for IFA using anti-HA, anti-SAG1 antibodies wherein the parasite number per parasitophorous vacuole was analyzed.

Electron microscopy

The TgLIPIN-ikD parasites were grown from 12 h and 24 h in the presence and absence of ATc, in labteks (Nunk, Thermofisher). The labteks containing parasite infected HFF were fixed in 0.1 M cacodylate buffer with 2.5% glutaraldehyde for 2 h and kept at 4 • C until further processing.

During processing, the sample were fixed again for 1 h with 1% osmium tetroxide in cacodylate buffer followed by overnight treatment in 2% uranyl acetate in distilled water. After dehydration in graded series of acetonitrile, samples were progressively impregnated in Epon812, the wells were then filled with fresh resin and allowed to polymerize 48 h at 60 • C. Ultrathin 70 nm section were obtained with a Leica UCT Ultramicrotome and collected on copper grids. Grids were poststained with uranyl acetate and lead citrate before their observation on a Jeol1200EXII

Transmission Electron Microscope. All chemicals were from Electron Microscopy Sciences.

Nile red staining of lipid droplets

The TgLIPIN-ikD parasites were allowed to infect and grow in confluent monolayer HFF grown on coverslips, in the +/-ATc conditions for 24 h and 48 h. Similar to IFA, these coverslips were fixed using 2.5% PFA, permeabilized with 0.25% triton X-100 and then stained with primary rat anti-HA antibody followed by detection with secondary AlexaFluor 488-conjugated goat anti-rat antibody. Thereafter, the sample coverslips were incubated for 1 h with nile red in 1X PBS before proceeding to DNA staining with Hoechst. The coverslips were mounted onto a glass slide in fluorogel prior to imaging using fluorescence microscope (Axio Imager 2_apotome; ZEISS). For visualizing nile red stained droplets yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) [START_REF] Greenspan | Nile red: A selective fluorescent stain for intracellular lipid droplets[END_REF] was used on the axio-imager. Quantification in +/-ATc condition was done by counting the no. of lipid droplets per parasite vacuole.

Heterologous complementation

Codon-optimized carboxy terminal lipin sequence (548-765 a.a) harboring the catalytic HAD domain (DVDGT), obtained from GenScript was ligated to NotI/MluI digested pD0170 yeast expression vector (obtained from Carman's lab). Cloned vector CLIP TgLIPIN-pD0170 was transformed into yeast strain ∆dpp1∆lpp1∆pah1 (kind gift from Dr. George Carman's lab, Rutgers

Center for Lipid Research, New Jersey ) and the transformants were screened on solid SD medium containing YNB agar lacking histidine, tryptophan, leucine, and uracil. Positive transformants were then proceeded for temperature sensitivity assay using drop-test. Briefly, the WT yeast strain, untransformed yeast strain ∆dpp1∆lpp1∆pah1 positive transformants from the plate were grown respectively in YPD and SD medium (Ura-, His-, Trp-and Leu-) to reach to O.D600 value 1. This culture was then diluted ten-fold, up to 5 times in a 96-well plate (first well is undiluted, 2 nd 10 -1 dilution, 3 rd 10 -2 dilution etc) and 5ul of each dilution was drop plated onto YPD agar plate. The plate was allowed to soak the yeast drop culture for up to 10-15 min prior to further incubation.

The experiment was performed in duplicate to be tested for growth at 30 • C and 37 • C separately for at least 48 h post inoculation.

Lipidomic analysis

The parasites were grown for 24 h ad 48 h in +/-ATc conditions within a confluent monolayer of HFF in flasks (175 cm 2 ). At each time point, parasites were harvested as intracellular tachyzoites

(1 × 10 7 cell equivalents per replicate) after syringe filtration with 3-μm pore size membrane.

These parasites were metabolically quenched by rapid chilling in a dry ice-ethanol slurry bath and then centrifuged down at 4 • C. The parasite pellet thus obtained was washed with ice-cold PBS thrice, before transferring the final pellet to a microcentrifuge tube. Then total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v) containing PC (C13:0/C13:0), 10 nmol and C21:0 (10 nmol) as internal standards for extraction. Next, the polar and apolar metabolites were separated by phase partitioning by adding chloroform and water to give the ratio of chloroform/methanol/water as 2:1:0.8 (v/v/v). For lipid analysis, the organic phase was dried under N2 gas and dissolved in 1-butanol to obtain 1µl butanol/10 7 parasites.

Total lipid analysis -The extracted total lipid sample was then added with 1 nmol pentadecanoic acid (C15:0) as internal standard and online derivatized to give fatty acid methyl ester (FAME) using trimethylsulfonium hydroxide (TMSH, Machenery Nagel) for total glycerolipid content.

Resultant FAMEs were then analyzed by GC-MS as previously described (Dubois et al. 2016). All

FAMEs were identified by comparison of retention time and mass spectra from GC-MS with authentic chemical standards. The concentration of FAMEs was quantified after initial normalization to different internal standards and finally to parasite number.

Free fatty acid and cholesterol analysis -Total lipid was dried and derivatized with BSTFA+TMCS, 99:1 (Sigma) to generate trimethylsilyl (TMS-) fatty acids and TMS-cholesterol.

These TMS derivatives were analyzed by GCMS as described above.

Phospholipid and neutral lipid analysis-For phospholipid analysis, the extracted total lipid extracted (as above) was separated with 1 nmol PA(C17:0/C17:0) (Avanti Polar lipids) by twodimensional silica gel high-performance thin layer chromatography (HPTLC, Merck). The solvent system used for the 1 st and 2 nd dimension was chloroform/methanol/28% ammonium hydroxide,12:7:1.6 (v/v) and chloroform/acetone/methanol/acetic acid/water, 10:4:2:2.6:1

(v/v/v/v/v)
, respectively. For DAG, TAG, Free fatty acids (FFA) and cholesteryl ester (CE) analysis, total lipid fraction was separated by 1D-HPTLC using hexane/diethyl ether/formic acid, 80:20:2 (v/v/v) as solvent system. Then each lipid spot on the HPTLC plate was scrapped off and lipids were methanolized with 200 μl 0.5 M methanolic HCl in the presence of 1 nmol pentadecanoic acid (C15:0) as internal standard at 85ºC for 3 h. The resulting FAMEs were extracted with hexane and analyzed by GC-MS (Agilent).

Stable isotope metabolic labelling experiment

Tracking FASII origin fatty acids -The TgLIPIN parasites were infected to confluent monolayer of HFF in glucose free-DMEM (1% FBS) supplemented with U-13 C-glucose or U- 12 C-glucose at a final concentration of 800 µM, with or without ATc (0.5 μg ml -1 ). The parasites were harvested up to 48 h post depletion of TgLIPIN and metabolically quenched as described previously (Amiar et al 2016). Lipid were extracted, derivatized using TMSH (Macherey-Nagel) and analyzed by GC-MS as described above. 13 C incorporation to each fatty acid was calculated as the percent of the metabolite pool containing one or more 13 C atoms after correction for natural abundance and the amount of 13 C-carbon source in the culture medium. The degree of the incorporation of 13 C into fatty acids (%carbon incorporation) was determined by the mass isotopomer distribution (MID) of each FAMEs. MID was obtained from the shift in isotopic mass dependent on the amount of 12 C carbons compared to the integration of 13 C carbon atoms. The total abundance of 13 Clabelled fatty acids was obtained by calculating the concentration of all isotopomers of 13 Clabelled FAMEs and finally normalizing to authentic internal standards and parasite number.

Tracking host-derived fatty acids -

The HFF cells were grown (1 × 10 8 cell equivalents per replicate) to confluency in the presence of stable isotope U- 13 C-glucose at a final concentration of 800 µM added to a glucose-free DMEM.

These 13 C-pre labelled HFF were then infected with TgLIPIN-iKD parasites in the presence of normal-glucose containing DMEM under +/-ATc (0.5 μg/ml). The host HFF and parasites were metabolically quenched separately, and their lipid content was quantified by GC-MS as described above. As described previously, the degree of the incorporation of 13 C into fatty acids (%carbon incorporation) is determined by the mass isotopomer distribution (MID) of each FAMEs. The total abundance of 13 C-labelled fatty acids was analyzed initially for HFF to check labelling of the metabolites (described previously). Later, the same was calculated for parasites to confirm direct uptake of 13 C-labelled fatty acids from the host.

Tracking uptake of deuterated fatty acid from medium (host environment)-Deuterated palmitic acid ( 31 d-C16:0) was dissolved in 10 mM in fatty acid-free bovine serum albumin/PBS solution by sonication in water bath for 30 min followed by incubation at 55ºC for 30 min. Freshly egressed TgLIPIN parasites were allowed to invade a confluent monolayer of HFF for at least 2 h under conditions of +/-ATc. Following invasion, the uninvaded parasites were washed off with DMEM and further allowed to grow in the normal culture medium-DMEM (1% FBS) containing 31 d-C16:0 at a final concentration of 0.1 mM in +/-ATc until 24 and 48 h of growth. The parasites were harvested by metabolic quenching as described previously. Lipid were extracted, derivatized using TMSH as well as TMS and further analyzed by GC-MS (described previously)

Statistical analysis for all experiments

Entire graphical data for this study was generated using GraphPad prism software. Three biological in total lipid from TgLIPIN-iKD+ATc or -ATc. i) 13 C-U-Glucose labelled fatty acids in the host cells. j) Significant increase in the oleic acid (C18:1) in the host derived 13 C labelled total fatty acids in the TgLIPIN-ikD+/-ATc. k)

replicates
Significant increase in host-derived 13 C labelled C16:0 and stearic acid (C18:0) in the free fatty acid pool in TgLIPIN-iKD+ATc. l) Excess 13 C-labelled C18:1 from the host was directly channeled to major membrane phospholipid within

TgLIPIN-ikD parasite upon ATc induction. Error bars = standard error of the mean, n=3 (no. of biological replicates). The major findings of this study are enlisted below: i. T. gondii genome encodes for 7 putative ACSs. These TgACSs possess canonical eukaryotic ACS motifs within their characteristic AMP-binding domain.

ii. The identified TgACSs localize to different sub-cellular compartments of the parasite, thereby suggesting their non-redundant roles in FA metabolism.

iii. TgACS3, is essential for parasite intracellular development and adaptation to different host nutritional conditions.

iv. Lipidomic data suggest that TgACS3 is potentially required for metabolic utilization of FAs for phospholipid biosynthesis within the parasite.

INTRODUCTION

Apicomplexan parasites are a group of unicellular eukaryotes that include pathogens responsible for various morbidity causing diseases. These include Toxoplasma gondii, Plasmodium sp. and

Cryptosporidium parvum causing the infectious diseases toxoplasmosis, malaria and cryptosporidiosis respectively. Eradicating these pathogens is a worldwide priority and the renewal of our therapeutic arsenal relies on understanding of the host-parasite interactions. Metabolic pathways that sustain parasite intracellular development and therefore host-parasite interactions represent ideal targets for drug development. One of the key determinants impacting the pathogenicity of these parasites within their host is lipid metabolism.

Toxoplasma gondii is capable of infecting virtually any nucleated cell, thereby displaying its metabolic strength in adapting to different nutritional environments. During the intracellular development of T. gondii, lipids play an essential role by providing structural building blocks for membrane biogenesis (Ramakrishnan et al. 2012;Amiar et al. 2016), as signaling molecules participating in key events like active invasion and egress through microneme secretion (Bullen et al. 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF]) and also as storage fuels, which plays critical role to maintain proper intracellular development [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]). To cope with the continuous need for lipids with the different host cells environments, T. gondii has evolved so it harbors three de novo synthetic pathways to generate fatty acids (FA), central lipid building blocks: (i) the apicoplast resident prokaryotic type-II fatty acid biosynthesis pathway (FASII) (Mazumdar and Striepen 2007;Amiar et al. 2016), (ii) the ER-based FA elongation pathway (Ramakrishnan et al. 2012) and (iii) the eukaryotic type-I fatty acid synthase present in the cytosol (Mazumdar and Striepen 2007).

Additionally, the parasite is also heavily reliant on acquiring FA directly from their hosts (Ramakrishnan et al. 2012;[START_REF] Fu | Synergistic Roles of Acyl-CoA Binding Protein (ACBP1) and Sterol Carrier Protein 2 (SCP2) in Toxoplasma Lipid Metabolism[END_REF][START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]Amiar et al. 2019;[START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]).

Ongoing research on parasite lipid metabolism suggests that the FA flux derived from both apicoplast FASII (Mazumdar et al. 2006;Amiar et al. 2016) as well as from direct scavenging from the host (Ramakrishnan et al. 2012;[START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF][START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]Amiar et al. 2019) are both essential for intracellular parasite viability. Furthermore, major phospholipid classes making the bulk of T. gondii lipid composition are obligate "patchwork molecules" composed of FA from the host and made de novo (Amiar et al. 2016). Taken together, FA, and most importantly their acquisition and utilization are pivotal pathways for parasite survival.

One of the key biochemical steps required for general FA utilization and trafficking is its activation by thioesterification to a Co-Enzyme A (CoA) that is catalyzed by the essential acyl-CoA synthetase (or acyl-CoA ligase) family [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). Biochemically, ACS catalyzes a twostep reaction to form fatty acyl-CoA. The initial step involves the formation of an adenylated intermediate through the hydrolysis of an ATP molecule, thus releasing pyrophosphate [START_REF] Black | Cloning, Sequencing, and Expression of the FadD Gene of Escherichia Coli Encoding Acyl Coenzyme A Synthetase[END_REF]). The ATP-activated enzyme then binds to the carboxyl group of incoming a free FA (FFA) moiety through an acyl bond to the phosphoryl group of AMP. Final fatty acyl-CoA product is formed after the transfer of the fatty acyl group to the sulfhydryl group of coenzyme A, thereby releasing AMP [START_REF] Black | Cloning, Sequencing, and Expression of the FadD Gene of Escherichia Coli Encoding Acyl Coenzyme A Synthetase[END_REF]).

Acyl-CoA synthases (ACS) exist as different isoforms in eukaryotes depending upon FA substrate specificity, which is based on FA chain length and degree of unsaturation. Humans have 26

enzymes that comprise the large ACS family activating short-chain, medium chain, long chain and very long chain fatty acid substrates [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). There are 9 long chain ACSs (LACS)

that have been well characterized in Arabidopsis thaliana (Shockey, Fulda, and Browse 2002).

This family of AMP-binding domain containing enzymes to a larger extend are indispensable for fatty acid metabolism in various eukaryotes. ACSs have been shown to be predominantly involved in glycerolipid biosynthesis as well as catabolic β-oxidation pathway and transport of fatty acids across organellar membranes [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF][START_REF] Zhao | Functional Overlap of Long-Chain Acyl-CoA Synthetases in Arabidopsis[END_REF].

The phylum apicomplexa is engaged actively in fatty acid metabolism and hence the role of the ACSs in parasite survival within its host cannot be undermined. ACSs in apicomplexa potentially activate fatty acids derived from both de novo synthetic machinery and from the host. This can be supported with the existence of ACS encoding genes in the genome of Cryptosporidium and Theileria which lack the de novo fatty acid biosynthetic machinery (Mazumdar and Striepen 2007).

Cryptosporidium parvum has been reported to have three AMP-binding domain containing longchain fatty acid CoA ligases out of which two CpACS1 and CpACS2 have been biochemically characterized as functional ACSs [START_REF] Guo | Differential Gene Expression and Protein Localization of Cryptosporidium Parvum Fatty Acyl-CoA Synthetase Isoforms[END_REF]. Plasmodium falciparum on the other hand has a large family of ACSs encoded by 13 genes (PfACS1a-PfACS12) [START_REF] Bethke | Duplication, Gene Conversion, and Genetic Diversity in the Species-Specific Acyl-CoA Synthetase Gene Family of Plasmodium Falciparum[END_REF]). Bethke et al also describes ACSs as the only family of metabolic enzymes that are encoded by genes expanded into the sub telomeric region which usually harbors parasite virulence genes [START_REF] Bethke | Duplication, gene conversion, and genetic diversity in the species-specific acyl-CoA synthetase gene family of Plasmodium falciparum[END_REF]. Drugs specific to ACSs in C. parvum have shown potent parasite killing activity, thereby hinting that this enzymatic reaction is a potential Achilles heel for apicomplexans [START_REF] Guo | Amelioration of Cryptosporidium Parvum Infection in Vitro and in Vivo by Targeting Parasite Fatty Acyl-Coenzyme a Synthetases[END_REF]).

Here, we identified 7 enzymes in Toxoplasma gondii which we classify as putative acyl-CoA synthases (TgACS). All the putative TgACSs were endogenously tagged and localize to nonoverlapping sub-cellular compartments of the parasite, including vesicular, mitochondrial, perinuclear-ER and basal end/cap of the tachyzoites. We further characterized two most interesting candidates, the mitochondrial TgACS2 and the cytosolic-perinuclear TgACS3 by generating inducible knockdown parasite lines using the classical Tet-off system. Downregulation of these

shows that TgACS2 has a minor role in maintaining parasite survival whereas TgACS3 is critical for tachyzoite intracellular development. TgACS3 is especially required for maintaining the division of large parasite vacuoles, with a marked role under high nutrient host environments.

Lipidomic analyses further show that the disruption of TgACS3 significantly reduces phospholipid content along with a concomitant and significant increase of free FA content within the parasite.

Our analysis therefore suggests that TgACS3 is the major source of FAs most likely obtained through host scavenging, which are then fueled to generate bulk phospholipid content for maintaining tachyzoite division.

RESULTS

Identification of seven genes encoding putative acyl-CoA synthetase (ACS) enzymes within the Toxoplasma genome

In order to identify putative members of ACS gene family in Toxoplasma gondii, we searched for genes with AMP binding domain in the ToxoDB database (http://toxodb.org/toxo/), and identified several candidate genes. Within these candidates bearing the AMP-binding domain, seven proteins EuPathDB (https://eupatdb.org). These homologs clustered together with TgACS7 with confident bootstrap scores forming a previously unidentified novel group of ACSs within apicomplexa [START_REF] Guo | Differential Gene Expression and Protein Localization of Cryptosporidium Parvum Fatty Acyl-CoA Synthetase Isoforms[END_REF]) (Fig. 2).

Putative TgACSs localize to non-overlapping intracellular compartments of T. gondii tachyzoites

To determine the physiological localization of the 7 putative TgACS, we generated T. gondii tachyzoite parasite lines expressing each protein fused to a C-terminal triple haemagglutinin (3×HA) epitope-tag under control of their endogenous promoter (Huynh and Carruthers 2009).

Immunofluorescence assays (IFA) using anti-HA antibody revealed that the 7 TgACSs localize to distinct sub-cellular compartments of the parasite (Fig. 3a). TgACS1, TgACS6 and TgACS7 were localized widely within the parasite cytoplasm, mostly as vesicular patterns (Fig. 3a). Interestingly, TgACS2 localized in a compartment reminiscent of the parasite mitochondria (Fig. 3a). Confocal microscopy and 3D reconstruction with the T. gondii mitochondrial outer membrane marker TOM40 ( [START_REF] Van Dooren | The import of proteins into the mitochondrion of toxoplasma gondii[END_REF]) confirmed the close vicinity and the co-localization of TgACS2

with the parasite mitochondria (Fig. 4a,b). TgACS3 was localized broadly at the cytosol, accumulating around and yet avoiding the nuclear area of the parasite (Fig. 3a). TgACS4 showed a distinct and interesting peri-nuclear/ER-like localization (Fig. 3a). Finally, TgACS5 displayed an interesting cytoplasmic localization, particularly enriched at the basal end of the parasite. (Fig. 3a). These localizations suggest non-redundant functions of ACSs within the different endomembrane compartments and cytoplasm of T. gondii. The ACSs in other eukaryotes have different fatty acid substrate specificities and functions depending upon their differential localizations [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF][START_REF] Fulda | Two Long-Chain Acyl-CoA Synthetases from Arabidopsis Thaliana Involved in Peroxisomal Fatty Acid β-Oxidation[END_REF][START_REF] Schnurr | Fatty Acid Export from the Chloroplast. Molecular Characterization of a Major Plastidial Acyl-Coenzyme A Synthetase from Arabidopsis[END_REF].

The endogenously tagged parasite lines were further confirmed by western blot analysis using anti-HA antibody (Fig. 3b). The migration of all the TgACS-HA was in accordance to their annotated protein sizes on ToxoDB (Fig. 3b). TgACS2-HA, however, showed a size shift of approximately 10 kDa below the predicted protein size (103 kDa), appearing at ~95 kDa (Fig. 4c).

To assess the importance of TgACSs, we probed their crispr based phenotype scores (Sidik et al. 2016) together with their endogenous localizations (Fig. 3a). Among all TgACSs only TgACS1 and TgACS3 displayed negative score of -1.88 and -2.53, respectively (Fig. 3a), suggesting their important/essential role for the intracellular development of T. gondii tachyzoites.

TgACS3 is critical for tachyzoite intracellular development especially in high host nutrient environments

Based on negative phenotype scores and an interesting mitochondrial localization putatively linked to adaptive metabolic function, we generated inducible knockdown tachyzoite strains for TgACS1, TgACS2, and TgACS3. The inducible knockdown lines (ikD) were generated by promoter replacement using the Tet-off system in TATi_ΔKu80 background (Meissner, Schlüter, and Soldati 2002;Sheiner et al. 2011) with an additional N-terminal HA-tagging (i.e. TgACS1/2/3-ikD-HA). Promoter replacement and insertion of the HA tag were confirmed by PCR (Fig. S2).

Intriguingly, although we obtained and recovered several polyclonal cell lines expressing TgACS1-iKD-HA but we were never able to obtain its monoclonal cell line, unlike TgACS2 and TgACS3.

Interestingly, the N-terminally tagged TgACS2-iKD-HA migrated approximately 10 kDa higher than the endogenously C-terminally tagged TgACS2-HA, with an apparent size of ~103kDa, correlating its predicted size on ToxoDB (Fig. 4c). Based on our dual tagging approach, we can assume that the C-TgACS2-HA is representative of correct protein size (~ 95 kDa) most likely due to its expression from endogenous promoter. It further suggests the existence of a probable different start codon for TgACS2 than the one annotated on ToxoDB.

Western blot and IFA analyses both confirmed the downregulation of respective proteins within

TgACS2-iKD-HA (Fig 4d,e) and TgACS3-iKD-HA (Fig. 5a,b) parasite lines by anhydrotetracycline (ATc) treatment. Complete protein depletion was observed at 24 h and 48 h after ATc treatment, respectively for TgACS2 (Fig. 4c) and TgACS3 (Fig. 5a). IFA confirmed the respective localization of TgACS2 at the parasite mitochondria (Fig. 4d, upper panel), and

TgACS3 at the cytosol/perinuclear compartment (Fig5b, upper panel). IFA also revealed that the ATc treatment leading to TgACS2 depletion did not affect the overall morphology of intracellular tachyzoites. On the other hand, the disruption of TgACS3 had a direct deleterious effect on tachyzoite intracellular morphology, especially on large vacuoles (>10parasites) where parasites appeared round and unhealthy (Fig. 5b, lower panel, phase).

We further performed plaque assays in the presence of different host nutritional environments (0,1 and 10% FBS containing growth media/DMEM) to probe the mutant's capacity to maintain normal growth under these conditions. We previously showed that wild type parasites sense host nutritional status and grow better with higher levels of host nutrient (10% FBS) (Amiar et al. 2019).

The disruption of TgACS2 had no effect on the intracellular development of tachyzoites at any levels of host nutrient (0%, 1% or 10% FBS) as shown by the absence of difference in plaque area also represented graphically (Fig. 4e,f). However, the disruption of TgACS3 had a negative impact on the parasite growth, which escalated with increasing levels of FBS in the growth culture medium. When grown in regular culture conditions with 1% FBS, parasites lacking TgACS3 had significantly smaller and lesser plaques, although it was only mildly reduced compared to the control (Fig. 5c,d). Importantly, when grown in high nutrient environment, at 10% FBS, parasites lacking TgACS3 displayed a much stronger growth phenotype than at 0 and 1% FBS, with significantly lesser and smaller plaques than in the control (Fig. 5c,d). Reduction of host nutrient to 0% FBS had no effect on growth of TgACS3-iKD in the +/-ATc (Fig. 5c,d). The growth phenotype was confirmed by a replication assay where TgACS3 depleted parasites had significantly fewer large vacuoles (7-10 parasites) and more smaller vacuoles (3-6 parasites) (Fig. 5e). TgACS3-iKD parasites displayed a mild egress defect in the presence of ATc (Fig. 5f). The egress assay was performed using calcium ionophore A23187 to chemically induce parasite egress.

Disruption of TgACS3 leads to reduction of parasite phospholipids and concomitant increase of free fatty acid content

Our results suggest that TgACS3 has an important role for the division, overall intracellular tachyzoite development and potential metabolic adaptation of the parasite to different host nutritional environments. To determine its precise function, we performed lipidomics analysis on

TgACS3-iKD +/-ATc (48 h time point) using gas chromatography-mass spectrometry (GC-MS)

approaches. We first determined the overall fatty acid (FA) abundance and composition of total lipid fraction (including FAs from all glycerolipids, neutral lipids and free FA). Overall, we did not observe any significant difference in the total lipid abundance of TgACS3-iKD (+ATc and -ATc) (Fig. 6a). However, the detailed profile of the total FA composition showed a slight yet significant increase in oleic acid (C18:1) content in the mutant (TgACS3-iKD +ATc) (Fig. 6b), possibly suggesting a substrate specificity of the enzyme for C18:1. To assess this and determine the precise function of TgACS3, total lipid was separated by high performance thin layer chromatography (HPTLC), and the composition and content of phospholipids (PL), free fatty acid (FFA), and storage lipid triacylglycerol (TAG) were quantified by GC-MS. Interestingly, this detailed lipidomic analysis revealed that the overall PL content was significantly reduced in the TgACS3-iKD +ATc mutant (Fig. 6c). Furthermore, FFA were significantly increased in the mutant (TgACS3-iKD +ATc) (Fig. 6d). This suggested that due to absence of ACS activity (TgACS3) within the parasite, inactivated FA could not be used for PL synthesis and thus accumulated as toxic FFA. The FFA excess in TgACS3-iKD mutant (+ATc) corroborated with the increased growth defect phenotype in the presence of 10% FBS (Fig. 5c). However, disruption of TgACS3 resulted in a slight increase (insignificant) the levels of TAGs (Fig. 6e). Quantification of the FA composition of the FFA fraction revealed significant decrease in palmitic acid (C16:0) and increase of stearic acid (C18:0) in parasite lacking TgACS3 (TgACS3-iKD +ATc) (Fig. 6g). Analysis of the FA composition from TAG in the TgACS-iKD mutant (+ATc), showed a similar trend in the variation of FA species, i.e. decrease in C16:0 increase C18:0 (Fig. 6h).

DISCUSSION

Acyl CoA synthetases catalyze a fundamental and limiting reaction in FA metabolism: the activation of free FA via thioesterification to coenzyme A (CoA). This key reaction allows the fatty acyl CoA intermediates to participate further in trafficking, assembly into complex lipids, post-translational modification of membrane proteins, and/or the catabolism of FAs via βoxidation [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF].

A unifying feature of acyl-CoA synthetase (ACS) enzymes and the ANL super-family that comprises ACS, Non-ribosomal peptide synthase adenylation domain and Luciferase enzymes [START_REF] Gulick | Conformational Dynamics in the Acyl-CoA Synthetases, Adenylation Domains of Non-Ribosomal Peptide Synthetases, and Firefly Luciferase[END_REF][START_REF] Ellis | Acyl-Coenzyme A Synthetases in Metabolic Control[END_REF], is the presence of AMP-binding domain as their catalytic domain [START_REF] Babbitt | Ancestry of the 4-Chlorobenzoate Dehalogenase: Analysis of Amino Acid Sequence Identities among Families of AcyhAdenyl Ligases, Enoyl-CoA Hydratases/Isomerases, and Acyl-CoA Thioesterases[END_REF]. All the 7 proteins that we identified as putative TgACSs possess the consensus amino acid sequences mostly as a part of their annotated AMP-binding domains (Fig. 1a,1b). The multiple sequence alignment of entire protein sequence of the TgACSs with other eukaryotic counterparts suggested an overall identity of up to 30-40% with high conservation in the AMPbinding domain (Fig. S1). Phylogenetic analysis of the identified TgACSs with known ACS from other eukaryotes clustered them to various clades, potentially based on their enzymatic substratespecificity and function (Fig. 2). The interesting separate sub-grouping of TgACS7 alongside its homologs from coccidia and chromerida may indicate a coccidian-photosynthetic origin of TgACS7 potentially lost during the evolution of haemosporidians (P. falciparum).

The existence of several ACSs in the Plasmodium and Toxoplasma genome are indicative of their unique and central functions for maintaining parasite survival. These parasites harbor extensively diverse range of FA varying in chain lengths and degree of saturation/unsaturation. Thus, it is not surprising that the parasites have a long list of ACS encoding genes. Similarly, there are 26 ACSs in humans, 11 of which are long-chain (ACSL) and very long chain acyl-CoA synthetases (ACSVL) [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). These ACSL and ACSVL have the specificity to activate fatty acid up to 16-22 carbons in length [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). The ACSLs, however typically activate the highly abundant fatty acids in nature including palmitate (C16:0) and oleate (C18:1). Others include, short-chain ACSs (ACSSs) that typically activate acetate, propionate, or butyrate and medium-chain ACSs (ACSMs) that activate fatty acids ranging in chain lengths from C6 to C10 [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). Lastly, the bubble gum-ACSs called ACSBG gene family (also known as lipidosin) that are capable of activating FAs of chain length 16-24 (Lopes-Marques et al. 2018).

The Toxoplasma candidates TgACS2,3,4 and 5 clustered within the long chain ACS group alongside the human long chain ACSLs and ACSVLs (Fig. 2) whereas TgACS6 was found with short FA chain specific ACSS, corroborating with its annotated function of propionyl CoA ligase (C3).

The diverse sub compartmental localization of putative Toxoplasma ACSs indicates nonredundancy and their highly specific roles in fatty acid activation. The correlation between protein localization and its function has been established with extended family of ACSs in humans [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]. For example, the role of endogenous ACSL1 in liver in neutral lipid synthesis and FA oxidation could correspond to its dual localization at the endoplasmic reticulum (ER) and mitochondria [START_REF] Li | Liver-Specific Loss of Long Chain Acyl-CoA Synthetase-1 Decreases Triacylglycerol Synthesis and β-Oxidation and Alters Phospholipid Fatty Acid Composition[END_REF]). Similarly, cardiac ACSL1 localized at mitochondria, has a significant contribution to FA β-oxidation [START_REF] Ellis | Acyl-Coenzyme A Synthetases in Metabolic Control[END_REF]). ACSL3, which has been found on lipid droplets and ER, participates in FA uptake and glycerolipid biosynthesis [START_REF] Poppelreuther | The N-Terminal Region of Acyl-CoA Synthetase 3 Is Essential for Both the Localization on Lipid Droplets and the Function in Fatty Acid Uptake[END_REF]. The localization of Cryptosporidium parvum CpACS1 at the apical end of cell-free sporozoites has been linked to FA biosynthesis required during process of invasion, and/or early stage development [START_REF] Guo | Differential Gene Expression and Protein Localization of Cryptosporidium Parvum Fatty Acyl-CoA Synthetase Isoforms[END_REF]. Similar polarization of TgACS5 at the basal end of the Toxoplasma tachyzoites could suggest its potential role in parasite cell division and cytokinesis [START_REF] Hu | Organizational Changes of the Daughter Basal Complex during the Parasite Replication of Toxoplasma Gondii[END_REF].

Additionally, the localization of the ACS could also direct the fate of the acyl-CoAs by allowing the ACSs to interact with different proteins involved in the direct downstream processing. For example, human ACSL1 coimmunoprecipitates with mitochondrial outer membrane proteins carnitine palmitoyl transferase 1a (CPT1a) and voltage-dependent anionic channel (VDAC) [START_REF] Lee | Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex[END_REF]. This proximity of ACSL1 to CPT1a functionally places it in the aspect of mitochondrial FA β-oxidation as CPT1a converts acyl-CoAs to mitochondria activated forms called acyl-carnitines [START_REF] Lee | Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex[END_REF]. Recent data including ours, provide evidence for the presence of an active acyl-CoA binding protein-2 (ACBP) that localizes to the mitochondria of the parasite (Amiar et al. 2019;[START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF]. We suggest that TgACS2 (mitochondrial vicinity) and

TgACBP2 are potentially coherently involved in FA activation and transport within the parasite mitochondria. However, whether they participate in mitochondrial membrane biogenesis and/or rather β-oxidation is yet to be determined.

Amongst several functions of the ACSs includes channeling of FA towards energy yielding process of β-oxidation [START_REF] Grevengoed | Acyl-CoA Metabolism and Partitioning[END_REF][START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF], Shockey et al 2002). The mouse ortholog of human hsACSBG1, called mBG1 localizing in the form of vesicles in close proximity to mitochondria of neuronal cells was shown to be involved in mitochondrial βoxidation of the long chain fatty acid palmitate [START_REF] Pei | The Acyl-CoA Synthetase 'Bubblegum' (Lipidosin)[END_REF]. Interestingly, based on phylogenetic analysis TgACS1 clusters together with the hsACSBG-gene family known to be involved in β-oxidation [START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF]). The presence of FA β-oxidation in apicomplexa has been a relatively unexplored. In eukaryotes, this energy yielding process compartmentalizes to mitochondria and vesicular organelles called peroxisomes. There is genomic and bioinformatic evidence for the presence of peroxisomes in T. gondii [START_REF] Kaasch | Targeting and Subcellular Localization of Toxoplasma Gondii Catalase. Identification of Peroxisomes in an Apicomplexan Parasite[END_REF][START_REF] Ding | Toxoplasma Gondii Catalase: Are There Peroxisomes in Toxoplasma[END_REF][START_REF] Moog | Genomic and Proteomic Evidence for the Presence of a Peroxisome in the Apicomplexan Parasite Toxoplasma Gondii and Other Coccidia[END_REF]. Proteins imported into the peroxisomal lumen bear two canonical targeting sequences-peroxisomal targeting sequence type 1 (PTS1), a C-terminal tripeptide with the consensus sequence [SAC]-[KRH]-[LM] and peroxisomal targeting sequence type 2 (PTS2), an N-terminal peptide comprised of the amino acids [START_REF] Moog | Genomic and Proteomic Evidence for the Presence of a Peroxisome in the Apicomplexan Parasite Toxoplasma Gondii and Other Coccidia[END_REF]). In A. thaliana LACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence whereas LACS7 inhabits both functional PTS1 as well as a PTS2 [START_REF] Fulda | Two Long-Chain Acyl-CoA Synthetases from Arabidopsis Thaliana Involved in Peroxisomal Fatty Acid β-Oxidation[END_REF]. This peroxisomal LACS activity has been shown to be essential for seedling growth [START_REF] Fulda | Peroxisomal Acyl-CoA Synthetase Activity Is Essential for Seedling Development in Arabidopsis Thaliana[END_REF]). Moog et al. provides bioinformatic evidence for the presence of classical PTSs in the protein factors potentially involved in β-oxidation in apicomplexan parasites. These proteins are subjected to stage specific expression supporting the existence of this process during the stage of sporulation or encystment within the parasites [START_REF] Moog | Genomic and Proteomic Evidence for the Presence of a Peroxisome in the Apicomplexan Parasite Toxoplasma Gondii and Other Coccidia[END_REF]). TgACS1 was one of the proteins defined within the "high confidence" PTS1-targeted peroxisomal proteome for T. gondii. Vesicular localization and predicted PTS1 at the C-terminus of TgACS1 strongly suggests its role in putative β-oxidation of FA in T. gondii. Co-localization studies with peroxisomal markers like catalase and lipidomics can provide further confirmation.

[RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X- [HQ]-[LAF]
Another functional aspect of ACSs is their involvement in FA transport across different organellar membrane compartments. Yeast ACSs Faa1p and Faa4p have been shown to function together with FA transport system (involving the transporter Fat1p) thereby linking import and activation of FA to further events in intracellular metabolism [START_REF] Faergeman | The Acyl-CoA Synthetases Encoded within FAA1 and FAA4 in Saccharomyces Cerevisiae Function as Components of the Fatty Acid Transport System Linking Import, Activation, and Intracellular Utilization[END_REF]. In E. coli, the FA import is dependent on both outer membrane bound transporter FadL as well as acyl-CoA synthetase FadD [START_REF] Black | Cloning, Sequencing, and Expression of the FadD Gene of Escherichia Coli Encoding Acyl Coenzyme A Synthetase[END_REF]. In yet another study on human ACSs, it was found that the N-terminal region of ACSL3 was responsible to FA uptake alongside its ER/LD localization [START_REF] Poppelreuther | The N-Terminal Region of Acyl-CoA Synthetase 3 Is Essential for Both the Localization on Lipid Droplets and the Function in Fatty Acid Uptake[END_REF]. The transport of FA towards cellular fate can also be linked to metabolic trapping due to vectorial acylation of these FA by the ACSs, acting as the driving force of uptake of free FA from the extracellular environment. Human ACSL6 is required for cellular retention of omega-3-docohexaneoic acid in the brain which in turn protects against neurodegenerative disorders [START_REF] Fernandez | Acyl-CoA Synthetase 6 Enriches the Neuroprotective Omega-3 Fatty Acid DHA in the Brain[END_REF]). The FA transporter function of ACSs in Toxoplasma can be elucidated through core lipidomics involving 13 C-Glc based fluxometrics.

In metabolically active hepatocyte cells Huh7, ACSL3 has been shown to specifically channel activated FA (esp. C18:1, oleic acid) into phosphatidylcholine. A siRNA mediated knockdown of ACSL3 decreases oleate incorporation to PC with concomitant rise in the levels of cellular-free oleic acid [START_REF] Yao | Long Chain Acyl-CoA Synthetase 3-Mediated Phosphatidylcholine Synthesis Is Required for Assembly of Very Low Density Lipoproteins in Human Hepatoma Huh7 Cells[END_REF]. Similar to this study, the measured decrease of the total levels of FA content derived from phospholipids in the TgACS3-iKD alongside concomitant increase of free FA both corroborate with the functional role of the enzyme (Fig. 6).Lipidomic analysis of TgACS3-iKD +ATc illustrated a gross effect on the concentration of long chain fatty acids esp.

C18:1. The short chain fatty acids, mostly apicoplast FASII derivatives like C12:0, C14:0, and C16:0 appeared to only slightly affected. This suggests that TgACS3 probably is a long chain fatty acyl-CoA ligase which thioesterifies long chain FA scavenged from the host (C18:1) rather than the apicoplast-FASII specific short chain fatty acids (C14:0 and C16:0). However, the source of FA substrate can be determined precisely from 13 C-Glc based flux analysis. Absence of human ACS, hsACSBG1, is linked to X-linked adrenoleukodystrophy (X-ALD) which is characterized by the presence of very long chain FAs in plasma and tissues causing neurodegeneration [START_REF] Jia | X-Linked Adrenoleukodystrophy: Role of Very Long-Chain Acyl-CoA Synthetases[END_REF]). The A. thaliana ∆lacs4∆lacs9 double mutant accumulates linoleic acid (C18:2) by up to 300% in leaf tissue due to abrogation of the corresponding acyl-CoA synthetase activity [START_REF] Jessen | Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis[END_REF].

The growth defect for the TgACS3-iKD was significantly more pronounced in 10% serum conditions. This observation can be explained by linking it to lipidomic data showing increase in the free FA content within TgACS3-iKD (+ATc). As per its expected function, due to loss of TgACS3 within the parasite there is a reduction in the metabolic utilization of fatty acids via thioesterification. In the presence of lipid enriched 10% serum DMEM, the parasites normally uptake more FAs, however, are unable to balance its utilization due to absence of an important acyl-CoA synthetase TgACS3.

Despite having a negative phenotype score on Toxoplasma genome wide CRISPR-Cas9 screen (Sidik et al. 2016), the measured growth defect of TgACS3-iKD in normal nutrient conditions at 1% FBS was not as pronounced as predicted. However, in the presence of high host nutritional environment, the phenotype of TgACS3-iKD (+ATc) was more pronounced, correlating reported phenotype scores. These differences might be explained by a certain level of redundancy by the other TgACS when TgACS3 is depleted. However, the replication assay and egress defect demonstrated the importance of TgACS3 for the parasite intracellular development. The existence of several genes encoding the ACSs in different eukaryotes has been rooted as the cause of certain redundancy amongst their functionality and substrate specificity. In A. thaliana, the LACS9 and LACS4 functionally overlap with another ER-specific isoform LACS8 [START_REF] Jessen | Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis[END_REF].

Another comprehensive study suggests that in plants cuticular wax biosynthesis majorly involves caninum (NCLIV_018500). First step involved the curation of these protein sequences. The protein sequences were aligned (Clustalomega) and then gaps were removed from the alignment.

Finally, the phylogenetic tree was constructed using the maximum likelihood method in the PhyML program. The default substitution model (WAG) was selected. Graphical representation and edition of the phylogenetic tree were performed with cladogram.

T. gondii strains and cultures

The parasite host cells human foreskin fibroblasts (HFF) were cultured using Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and 25 μg/mL gentamicin (Gibco) at 37°C and 5% CO2.

T.gondii tachyzoite parental strains RH-ΔKu80 TATi, RH-ΔKu80 as well as mutant strains

TgLIPIN-iKD, TgLIPIN-3*HA were propagated by serial passage within their host HFF using DMEM supplemented with 1% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and 25 μg/mL gentamicin (Gibco) at 37°C and 5% CO2.

Generation of HA-tagged lines for all TgACSs and inducible knockdown line for TgACS2 and TgACS3

All C-terminally tagged HA expressing parasite lines were generated from gene's endogenous locus using the classical pLIC strategy using homologous recombination in a RH-ΔKu80 strain.

For the same, homology regions (HR) (respective lengths enlisted below: Table1) of all the TgACSs located towards to C-terminus excluding the stop codon, were amplified from the parasite genomic DNA using the primers enlisted in Table 1. The homology region was assembled into PacI digested pLIC-HA-DHFR plasmid using ligation independent cloning protocol. The assembled plasmid was linearized using single enzyme site specific to the parasite DNA sequence within the plasmid just before transfection (enlisted in table 1). Parasites were selected with the

Immunofluorescence assay

Primary antibodies anti-HA (Rat, Roche), anti-IMC1 (Mouse), anti-SAG1 (Mouse) were used at dilutions 1:500, 1:1000 and 1:1000 respectively. Secondary Alexa Fluor 488-and 546-conjugated anti-mouse, anti-rat and anti-rabbit antibodies (Life Technologies) were used at 1/2500. For the immunofluorescence assay (IFA) parasites were grown on confluent HFF on coverslips and fixed in PBS containing 2.5% paraformaldehyde (PFA) for 15 min at room temperature (RT). Samples were permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT prior to blocking in PBS containing 3% BSA and subsequent incubation with primary antibodies then secondary antibodies diluted in the blocking solution. Labelled parasites were stained with Hoechst (1/10000, Life technologies) for 20 min and then washed three times in PBS before final mounting of the coverslips on a glass slide using fluorogel (reference). The fluorescence was visualized using fluorescence microscope (Axio Imager 2_apotome; ZEISS).

Confocal Microscopy and 3D reconstruction

Confocal microscopy was performed for TgACS2-HA co-localized with parasite specific mitochondrial marker (TOM40), to delineate the proximity between the two proteins. For image processing, 3D reconstruction was achieved with the UCSF ChimeraX software from the raw data (i.e., TgACS2-HA co-loc with mitochondrial TOM40) obtained after initial processing i.e. by cropping the region of interest and then applying the ''Iterative Deconvolve 3D'' plugin for each channel, separately.

Western blot analysis

Parasites were harvested for western blot after complete egress from their host. In order to remove any host cell debris, the parasites were passed through a 3 μm filter, then counted by hemocytometer and solubilized in SDS buffer at equivalent cell densities. Equal amount of protein was separated on a 4-12% gradient SDS-polyacrylamide (Life Technologies) and transferred to

Nitrocellulose membrane (check this) using the XCellII Blot Module (Invitrogen). Primary antibodies anti-HA (Rat, Roche) and anti-TOM40 (Rabbit) ( [START_REF] Dooren | Toxoplasma Gondii Tic20 Is Essential for Apicoplast Protein Import[END_REF]) were used at a dilution of 1:500 and 1:1000, respectively. Secondary goat anti-mouse and anti-rabbit horse radish peroxidase (HRP) conjugated antibodies (Thermo Scientific) were used at 1:2000. Protein signal was detected by chemiluminescence after membrane staining with Luminata Crescendo

Western HRP detection kit (Millipore). The signal strength of protein was quantified using a BioRad Chemidoc imager (BioRad).

Phenotypic analysis

Plaque assay-The extracellular parasites were harvested after filtration and counted by hemocytometer. Then approx. 500 parasites were inoculated to confluent HFF flask (25 cm 2 ). The mutant parasites TgAC2-iKD and TgACS3-ikD was grown for plaque assay in the presence or absence of ATc (0.5 μg ml -1 ) for 7-10 days. Plaque sizes were visualized by crystal violet staining (30-60 min) after aspiration of culture media, and cells fixation with 100% ethanol (5 min) followed by phosphate-buffered saline (PBS) wash.

Replication assay-The parasites were grown for two days with or without ATc (0.5 μg ml -1 ), harvested and filtered. Equal number of parasites were allowed to invade confluent HFF grown on coverslips. Following 2 h of invasion, the coverslips were washed thrice with ED1 (1% FBS containing DMEM), in order to remove any uninvaded parasites and promote synchronized replication. Anhydrotetracycline (ATc) (0.5 μg ml -1 ) was added at the outset of the experiment, allowing the treatment for 24 h, alongside control parasites without ATc. These coverslips were then fixed and processed for IFA wherein the parasite number per parasitophorous vacuole was analyzed.

Egress assay-Like replication assay, the parasites were treated with ATc prior to the experiment.

Equal number of parasites were allowed to invade confluent HFF grown on coverslips and allowed to grow for 24-30 h for having big vacuoles with 16-32 parasites. For the egress assay, the culture media (ED1) was removed and replaced with either 5 µM of calcium ionophore A23187 or DMSO control. This was followed by a 4 min incubation at 37ºC. The parasites were immediately fixed with 2.5% paraformaldehyde+0.02% glutaraldehyde and proceeded for an IFA using anti-SAG1

antibody.

Lipidomic analysis

The parasites were grown for 24 h ad 48 h in conditions of +/-ATc on the confluent monolayer of HFF in flasks (175 cm 2 ). At each time point, parasites were harvested as the intracellular tachyzoites (1 × 10 7 cell equivalents per replicate) after syringe filtration with 3-μm pore size membrane. These parasites were metabolically quenched by rapid chilling in a dry ice-ethanol slurry bath and then centrifuged down at 4 • C. The parasite pellet thus obtained was washed with ice-cold PBS thrice, before transferring the final pellet to a microcentrifuge tube. Then total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v) containing PC (C13:0/C13:0), 10 nmol and C21:0 (10 nmol) as internal standards for extraction) for 1 h at 4 • C, with periodic sonication.

Then polar and apolar metabolites were separated by phase partitioning by adding chloroform and water to give the ratio of Chloroform/Methanol/Water, 2:1:0.8 (v/v/v). For lipid analysis, the organic phase was dried under N2 gas and dissolved in 1-butanol to obtain1µl butanol/10 7 parasites.

Total lipid analysis -Total lipid was then added with 1 nmol pentadecanoic acid (C15:0) as internal standard and derivatized to give fatty acid methyl ester (FAME) using trimethylsulfonium hydroxide (TMSH, Machenery Nagel) for total glycerolipid content. Resultant FAMEs were then analyzed by GC-MS as previously described (Dubois et al. 2018). All FAME were identified by comparison of retention time and mass spectra from GC-MS with authentic chemical standards.

The concentration of FAMEs was quantified after initial normalization to different internal standards and finally to parasite number.

Phospholipid and neutral lipid analysis-For phospholipid analysis, total lipid extracted (as mentioned above) was separated with 1 nmol PA(C17:0/C17:0) (Avanti Polar lipids) by twodimensional silica gel high-performance thin layer chromatography (HPTLC, Merck). The solvent system used for the 1 st and 2 nd dimension was chloroform/methanol/28% ammonium hydroxide,12:7:1.6 (v/v) and chloroform/acetone/methanol/acetic acid/water, 10:4:2:2.6:1 (v/v/v/v/v), respectively. DAG, TAG, Free fatty acids (FFA) and cholesteryl ester (CE) analysis, total lipid fraction was separated by 1D-HPTLC using hexane/diethyl ether/formic acid, 80:20:2 (v/v/v) as solvent system. Thereafter, each lipid spot on the HPTLC plate was scrapped off and lipids were methanolized with 200 μl 0.5 M methanolic HCl in the presence of 1 nmol pentadecanoic acid (C15:0) as internal standard at 85ºC for 3 h. The resulting FAMEs were extracted with hexane and finally analyzed by GC-MS (Agilent).

Statistical analysis for all experiments

Entire graphical data for this study was generated using GraphPad prism software. Three biological replicates were used per experiment (n=3). The error bars are representative of standard error of mean (SEM) for each study. Statistical significance was determined for each experiment by t-test using GraphPad Prism. Range of statistical significance was signified as per the p value, wherein 0.01-0.05=*, 0.01-0.001=** and <0.001=***.

Fig.2. Phylogenetic analysis of the seven TgACSs with eukaryotic homologs

All 7 TgACSs appear in specified clusters based on their homology with their eukaryotic counterparts: TgACS1 in ACBG cluster; TgACS2, TgACS3, TgACS4, TgACS5 in the long chain ACS group; TgACS6 in the short chain ACS cluster; TgACS7 in a separate cluster specific to coccidia and chromerids. The phylogenetic tree was generated online using Phylogeny.fr. HA antibody shows downregulation of TgACS2 24 after induction with anhydrotetracycline (ATc). f) Plaque assays performed with TgACS2-iKD depict normal growth after depletion of TgACS2 using ATc, with no significant effect in the presence of 0, 1 and 10% FBS conditions. g) Graphical representation and statistical analysis of the plaque areas obtained confirms the dispensability of TgACS2 for parasite growth. f) The FA composition of phospholipids in the TgACS3-ikD represented by mol%. Graphical representation of FA composition (mol%) of the FFA (g) and TAG (h) species within TgACS3-iKD. TgACS3-iKD+ATc in red and TgACS3-iKD-ATc in grey. g) TgACS3-iKD+ATc parasites were characterized with a significant decrease in C16:0 and increase in C18:0 derived from the FFA species. Three biological replicates were used per experiment (n=3). The The survival of apicomplexan parasites within their hosts is predominantly dependent on lipids, which play an essential role by regulating metabolic flux, acting as signaling molecules, storage fuels and structural building blocks of membranes. To sustain rapid replication during the intracellular stages, these parasites rely on active lipid synthesis fed by fatty acids derived from both de novo FASII and the host. The essential mechanism allowing these parasites to maintain a proper flux between the two sources of FA remains largely unknown. In pursuit of the same, my PhD focused on ascertaining pivotal roles of 2 key proteins in parasite FA metabolism: a) acyl coA synthetases (ACS) converting free FA to activated acyl coA-FA and b) Lipin: a phosphatidic acid phosphatase utilizing the activated FA to generate and control the bulk synthesis of central lipid precursors, phosphatidic acid (PA) and diacylglycerol (DAG).

TgLIPIN: key enzyme regulating FA fluxes via PA metabolism in Toxoplasma

Functional characterization of TgLIPIN established its pivotal function as an active phosphatidate phosphatase, regulating the critical levels of PA through the generation of its enzymatic product DAG. Fatty acid scavenging from the host can only support the proper intracellular growth of Toxoplasma tachyzoites in specific amount below a certain lipotoxic threshold [START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF]. This suggests that the parasite is reliant on a delicate tuning allowing these FA to be properly repurposed for metabolic use rather than accumulating in excess resulting in parasite growth arrest.

The parasite and the host work in a concerted manner to regulate and maintain the FA flux/homeostasis. We propose that TgLIPIN is an essential control switch that channels the hostderived FA flux to either membrane phospholipids or storage lipids (TAG). The role of PA in parasite pathogenicity has previously been attributed to (i) its function as a signaling lipid mediating key parasite processes like invasion, motility and egress (Bullen et al., 2016;Bisio et al., 2019), (b) as central precursor for generation of membrane phospholipids and regulator of LPA/PA levels which governs parasite cell division and cytokinesis (Amiar et al., 2016(Amiar et al., , 2019;;Shears et al., 2017). We, through this study, determined the unprecedented and key role of PA as the limiting component between glycerolipid synthesis and storage pathways in apicomplexa, particularly in T. gondii.

Identifying the protein regulators of the metabolic tap, TgLIPIN

In other eukaryotic systems, lipin functionality has been proposed to be under control of multiregulatory mechanisms involving various protein kinases and phosphatases (Zhang and Reue, 2017;Carman and Han, 2019). Data from our collaborative studies involving phospho-proteomic screen of a cyclin-dependent protein kinase-TgCDPK7 knockout strain (personal communication C. Botté, collaboration Botté-Sharma) showed that TgLIPIN was hypophosphorylated at serine residue in the TgCDPK7-KO strain (amino acid position-serine 1019). Interestingly, the phosphorylation of the yeast lipin Pah1 that controls its localization, catalytic activity and stability, is regulated by several protein kinases including cyclin dependent protein kinases, protein kinase A, protein kinase C (Carman and Han, 2019). Together, these data orientated my PhD project further towards the identification of potential regulators of TgLIPIN. In contrast to multi-site phosphorylation events governing the regulation of eukaryotic homologs of TgLIPIN, lipin-1 (in mammals) and Pah1 (in yeast), are activated by a single-site dephosphorylation event [START_REF] Kim | A conserved phosphatase cascade that regulates nuclear membrane biogenesis[END_REF][START_REF] Pascual | Phosphatidate phosphatase, a key regulator of lipid homeostasis[END_REF]Reue and Wang, 2019). This activating dephosphorylation is controlled by a specific protein phosphatase family: The Dullard protein phosphatase for the human lipin-1 and the related Nem1p (part of the Nem1p-Spo7p complex) for the yeast Pah1.

Using bioinformatic tools I identified a homolog of the Dullard/Nem1p phosphatase in T. gondii, which was named as TgDULL. We think that could be a putative regulator of TgLIPIN metabolic function. This Nem1p/Dullard homolog is predicted to be a protein phosphatase sharing the characteristic HAD motif with its potential/probable substrate TgLIPIN. I endogenously tagged TgDULL at its C-terminus with 3×HA tag. The corresponding TgDULL-HA was found colocalizing with the ER of the parasite placing it into the perspective of ER vicinity localization of TgLIPIN. I generated an inducible knockdown line for this protein, within which TgLIPIN will be endogenously tagged. The future aim and continuation of my PhD project is to determine the effect of TgDULL downregulation on localization and phosphorylation status of TgLIPIN. Subsequently, parasite development and survival will be sought to establish TgDULL role as the potential 'activator' of TgLIPIN.

Does the presence of a predicted Nuclear Localizing Signal, NLS, in Plasmodium falciparum

Lipin (PfLIPIN) mean that PfLIPIN has retained a link to phospholipid gene regulation like its human homolog lipin-1?

Another important future perspective of my lipin project is to complete the functional characterization of PfLIPIN. In contrast to TgLIPIN, PfLIPIN, was found to have a predicted nuclear localization signal (NLS) like its other eukaryotic counterparts (Csaki and Reue, 2010;[START_REF] Ren | A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis[END_REF][START_REF] Han | Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis[END_REF][START_REF] Carman | Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase[END_REF]. Although PfLIPIN is predicted to be mutable via the P. falciparum whole genome KO screen [START_REF] Zhang | Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis[END_REF], an intriguing localization of this protein in the vicinity/near overlap with the parasite nucleus, instils a potentially unexplored and important role. In humans, lipin-1 translocates to the nucleus to function as an inducible amplifier of PGC1 alpha/PPAR alpha pathway in hepatocytes to control the hepatic lipid metabolism (Finck et al., 2006). Another astounding study by [START_REF] Romanauska | The Inner Nuclear Membrane Is a Metabolically Active Territory that Generates Nuclear Lipid Droplets[END_REF], showed that the inner nuclear membrane (INM) of a eukaryotic cell is a metabolically active site that can, generate nuclear lipid droplets (nLDs). They examined the INM lipid composition and found that the INM was enriched with DAG and that the nLDs lipid monolayer comprised appropriate levels of PA/DAG controlling the cell's storage vs growth functions. This fine balance between PA and DAG is notably regulated via the nuclear activity of the lipin homolog in yeast, Pah1. Thus, the predicted NLS and interesting localization, could place PfLIPIN in the perspective of a possible link between nuclear metabolic activity and gene regulation in P. falciparum.

TgACSs: Role of the fatty acid 'activating' enzymes in Toxoplasma lipid metabolism

Acyl CoA syntheses (ACS) are enzymes that catalyze the initial and mandatory reaction of all FA metabolic pathways. Within apicomplexa, 13 ACSs have been identified in P. falciparum and 3 in C. parvum [START_REF] Bethke | Duplication, Gene Conversion, and Genetic Diversity in the Species-Specific Acyl-CoA Synthetase Gene Family of Plasmodium Falciparum[END_REF][START_REF] Guo | Differential Gene Expression and Protein Localization of Cryptosporidium Parvum Fatty Acyl-CoA Synthetase Isoforms[END_REF]. The extension of the PfACS9 into a family into 10 paralogs (PfAcs1a-PfAcs8) within the sub-telomeric region of the chromosomes, which is otherwise well known for harboring parasite virulence specific 'var/rifin' genes, strongly confer that lipid metabolism in important for the pathogenicity of the parasite [START_REF] Bethke | Duplication, Gene Conversion, and Genetic Diversity in the Species-Specific Acyl-CoA Synthetase Gene Family of Plasmodium Falciparum[END_REF].

With the help of bioinformatic and genetic tools, we were able to identify 7 putative acyl CoA synthetases in T. gondii. Phylogeny helped segregate the 7 ACSs with their closest eukaryotic counterparts with an interesting separation of TgACS7 to sub-group specific to coccidia and chromerids. This protein could be an evolutionary link between apicomplexan sub-group coccidia and plant-like ancestors, chromerids, which was lost in P. falciparum.

Characterization and lipidomics on mutant parasites lacking the predicted most important member of the Toxoplasma ACS gene family, TgACS3, provided insights into the functional aspect and importance of the protein family for the parasite. First, the increase in FFA species with simultaneous decrease in the FA levels of the phospholipid classes within this mutant parasite (TgACS3-iKD), strongly supported its functional aspect of being an ACS. Importantly, it determines its likely role for activating FA for their downstream metabolic flux for phospholipid synthesis, membrane biogenesis and parasite division.

Functional and metabolic confirmation of all TgACSs may require further biochemical assays involving activity assay using purified recombinant TgACS and/ or heterologous complementation of appropriate yeast strains lacking ACS activity. Such assays have been reported previously (Shockey, Fulda and Browse, 2002). We have initiated this work, but I was unfortunately not able to fully conduct and report it in this manuscript. To further determine the functional role of the remaining TgACS, we are currently generating CRISPR-cas9 mediated knockout strains for each ACS identified in T. gondii genome. One of the future prospects of my PhD project is thus to assert the effect of the disruption of all TgACSs on parasite growth/fitness of the parasite, which will be followed by core lipidomics to ascertain their FA specificities and role for parasite lipid synthesis.

It is interesting to note that of all the ACSs that we identified in Toxoplasma, none was localizing to one of the major metabolic hubs, apicoplast. Despite being highly active in PA biosynthesis, the absence of ACS suggests that the substrates utilized in this process are 'acyl-ACPs' directly obtained from FASII rather than fatty acyl-CoA thioesters.

ACSs also participate in the vectorial acylation of FAs, a process coupling the import and activation of FA. An evidence of physical interaction between an ACS, FACS and an acyl-CoA transporter, Fat1p, in yeast, also indicates vectorial esterification and metabolic trapping of the FAs [START_REF] Zou | Vectorial acylation in Saccharomyces cerevisiae: Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex[END_REF]. A PVM specific ACS in C. parvum, CpACS1, has been proposed to activate FA acquired from the host, in order to facilitate their entry into the parasite cytoplasm. Thus, it is plausible that some of these TgACSs could participate in vectorial acylation of FAs. The mitochondrial localization of TgACS2 and TgACBP2 suggest their potential participation in vectorial acylation of fatty acids across the mitochondrial membrane. It is probably the case with other ACSs, ACBPs, and potentially other (lyso)phospholipases at other key metabolic active sites for parasite lipid synthesis. The results of my project will potentially help answering such questions on the metabolic organization of the key parasite enzymes that maintain lipid synthesis and homeostasis.

Lastly, FA flux analysis using stable isotope labelling with 13 C-Glucose, including our novel lipidomic approach to monitor host scavenging, will help determine the role of ACS at the cross-roads of utilization of FA from the two key parasite sources: de novo machinery and scavenging from host. Determining these will be key to unravel the complex mechanisms sustaining lipid synthesis and parasite propagation and survival.

Understanding the bigger perspective: Metabolic co-evolution of the host with its parasite By nature of its intracellular development parasite metabolism cannot be studied exclusive of the host. As a part of their survival regime, these parasites hijack their host cell machinery to acquire nutrients and evade immune response. These parasites infect multiple host cell types and are able to establish successful infection owing to consistent remodeling within the metabolic capacities.

Recent study showed that malaria infection in humans is potentially impacted by the host dietary calorie alterations (Mancio-Silva et al., 2017). Use of restricted diet regime in mouse models prior to infection with P. berghei resulted in attenuated parasitemia and virulence. The basis of this nutrient sensing was identified as a homolog of SNF1/AMPKα, a putative serine/threonine kinase called KIN which modulated the transcription of genes in response to host nutritional status. The calorie restriction mediated effects on parasite replication within the host were in fact rescued by addition of glucose. This shows the impact of host nutritional environment on parasite metabolic characteristics which in turn impacts their infection rate within the host. An interesting epidemiological study of African ethnic tribes revealed natural resistance of Fulani tribe to malaria.

The basis on this divergence has been long rooted into genetic polymorphisms and immune responses. However, it is interesting to note that these resistant tribes also diverge in their dietary habits which involves intake of food rich in saturated fatty acids. Although, there is no evidence of direct link between resistance to malaria infection and food intake, it plausible to co-relate the dietary intake to host nutritional environment affecting parasite infection (Zuzarte-Luís and Mota, 2018). The sexual stages of P. falciparum respond to levels of host derived lysophosphatidylcholine (LPC). High levels of LPC suppresses the development of gametocytes in vitro [START_REF] Brancucci | Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum[END_REF]Wein et al., 2018). Consistently, the deprivation of lipids from growth culture media resulted in decline in the growth of otherwise unessential FASII-FabI knockout asexual blood stage P. falciparum parasites (Amiar et al., 2019). When subjected to weak host nutritional environment, the Toxoplasma tachyzoites are able to suffice their lipidic needs by upregulation of fatty acid synthesis de novo via FASII (Amiar et al., 2019). It can thus be said that the parasites endure adept metabolic capacity to ensure survival in less-favorable metabolic landscape.

The sexual development of Toxoplasma is restricted to feline intestine due to the excessive presence of linoleic acid (18:2) owing to the absence of the enzyme ∆desaturase responsible for metabolism of 18:2 (Di Genova et al., 2019). This study represents a classic example of coevolution of the host and parasite based on metabolic reprogramming, which in this case occurs within the host. As a part of its innate defence mechanism, the host, embarks a competitive streak by limiting the parasites from fatty acid uptake by enwrapping their PV in host mitochondria [START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma Gondii by Limiting Its Uptake of Fatty Acids[END_REF]. Host cells infected with T. gondii exhibit enhanced lipid droplet biogenesis due to increased levels of neutral lipid TAGs (Hu, Binns and Reese, 2017). Later, it was shown by Nolan et al that indeed the parasites acquire the host lipids through host Rab7 labelled vesicles/LDs originating from host Golgi to the parasite PV lumen (Nolan, Romano and Coppens, 2017).

Lessons from other apicomplexan parasites: De novo synthesis vs scavenging

So far, this thesis has extensively discussed the fatty acid biosynthesis and host/environmental scavenging in two sub-groups of apicomplexa, Coccidia (with ref to T. gondii) and Haemosporidia with ref to (P. falciparum). Further ahead, I briefly discuss the fatty acid metabolism in other apicomplexan parasites: a) Piroplasmida These livestock affecting parasites reside within a vertebrate host (as sporozoite and merozoite forms) and in their tick host (as zygote and ookinete form). Once inside the mammalian host, these protists sequentially parasitize nucleated lymphocytes followed by enucleated erythrocytes. This part of the life cycle bears resemblance to an extent with Haemosporidia (Plasmodium falciparum). An interesting aspect of the survival of these parasites within their lymphocyte host is that post 15 min of invasion and establishment of infection, the parasite escapes from the enclosing host cell membrane to lie freely in the host cytoplasm, surrounded by host microtubules [START_REF] Shaw | Cell Invasion by Theileria Sporozoites[END_REF]. Thus, during its intracellular development, this parasite is not surrounded by any parasitophorous vacuolar membrane. There are studies suggesting that these intracellular free parasite forms phagocytose or pinocytose the host cytoplasm [START_REF] Jalovecka | The complexity of piroplasms life cycles[END_REF].

As a part of their eubacterial ancestry, the piroplasms have retained organelle apicoplast, however its importance as the central metabolic hub still holds many questions. For instance, despite the presence of apicoplast, Theileria annulata lacks the enzymes involved in heme and fatty acid synthesis. No enoyl ACP reductase or acetyl-CoA carboxylase is apparent in the Theileria genomes [START_REF] Goodman | Fatty Acid Biosynthesis as a Drug Target in Apicomplexan Parasites[END_REF]. Similarly, despite the presence of an ACP in Babesia bovis apicoplast, the FASII pathway has been shown to be dispensable by triclosan treatment, during intraerythrocytic stages [START_REF] Caballero | Characterization of Acyl Carrier Protein and LytB in Babesia Bovis Apicoplast[END_REF].

The indispensability and/or absence of key enzymes involved in FASII pathway in the piroplasms residing within mammalian hosts suggests the complete dependency of these parasites on their host for fatty acids and other essential nutrients. This explains the 'nobarrier' nature of its intracellular development i.e. the complete absence of PVM to facilitate maximum uptake from the host. Nevertheless, it would be interesting to identify the nutrient transporters that actively participate in this the process of a 'regulated' uptake from the host cell in these parasites.

A study distinguishing the lipid composition of B. bovis infected erythrocytes from uninfected RBCs showed trend similar to P. falciparum with an increase in lipid species-PC, PA, DAG , CE. This study also showed by uptake studies that Babesia was able to scavenge resources including choline, myo-inositol and 14 C labelled stearic acid from the extracellular environment [START_REF] Jalovecka | Phosphatidylcholine Formation Is the Predominant Lipid Biosynthetic Event in the Hemoparasite Babesia Bovis[END_REF].

b) Cryptosporidida

This parasite completes both its asexual and sexual part of the lifecycle within epithelial cells lining the intestines of its human host. The parasites belonging to this sub-group have developed a unique epicellular lifestyle wherein they develop intracellularly within a parasitophorous vacuole bulging out of the apical end of cytoplasm of the host cell [START_REF] Elliott | Cryptosporidium Parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface[END_REF].

Biochemical and genetic data suggests that Cryptosporidium has lost a plastid as well as the genomes of both plastid and mitochondria. Subsequently, Cryptosporidium is unable to de novo synthesize amino acids, nucleotides and fatty acids [START_REF] Cacciò | Cryptosporidium: Parasite and Disease[END_REF].

Due to the absence of several de novo synthesis pathways, a considerable amount of the metabolites is up taken from host and or extracellular environment. Cryptosporidium has been known to have several transporters specific to each metabolite like amino acids (~11transporters), sugars (~9), and nucleotides (at least one). These parasites are also known to have approx. 24 ABC transporters [START_REF] Cacciò | Cryptosporidium: Parasite and Disease[END_REF].

Despite extensive reliance on host FA scavenging due to absence of apicoplast FASII, Cryptosporidium possesses a type I intron less fatty acid mega synthase which is similar to bacterial polyketide synthase. The specificity of the CpFASI for very long chain fatty acids (>C20) suggests that rather than de novo synthesis, this enzyme is potentially rewired to function as a fatty acid elongase. Apart from the CpFASI, these parasites also possess a membrane protein called LCE i.e long chain elongase and a bacterial like polyketide synthase (CpPKS1). The LCE enzyme accepts acyl CoA thioesters as its substrate rather than acyl-ACPs. CpLCE1 is able to elongate its substrate fatty acyl CoA ester by a single C2 subunit and was shown to have a preference towards myristyl CoA and palmityl CoA. This enzyme is present on the plasma membrane of sporozoites as well as in the PVM, to utilise the incoming scavenged fatty acids which prior to, are activated to corresponding thioesters by acyl-CoA synthetases (also localised towards the parasite's PM and PVM). It seems that all the plasma membrane bound important enzymes like ACS, ACBP and LCE are purposed towards these locations in order to achieve proper uptake of fatty acids via transport, activation and further elongation.

Based on simplified metabolic features and a compact genome, the Cryptosporidida can be placed phylogenetically closer to Gregarines rather than other coccidians and haemosporidians.

c) Gregarinida

Parasites belonging to Gregarinida and Cryptosporidida likely share a monophyletic relationship, meaning they may a common immediate ancestor. However, Gregarines are able to parasitize only invertebrates. Like Cryptosporidium, the Gregarina also lacks a plastid but in contrast possesses a respiring mitochondrion. Genome analysis of Ascogregarina sp hints at de novo fatty acid biosynthesis due to the retention of wide set of fatty acid (FA) biosynthetic enzymes including a type I fatty acid synthase, FA desaturases and fatty acid CoA ligases [START_REF] Templeton | A Genome-Sequence Survey for Ascogregarina Taiwanensis Supports Evolutionary Affiliation but Metabolic Diversity between a Gregarine and Cryptosporidium[END_REF].

Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling

SUMMARY

Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso) phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wildtype parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant ( 1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and ( 2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite.

INTRODUCTION

Apicomplexa are intracellular protozoan parasites that cause serious infectious diseases in humans, including malaria and toxoplasmosis. Most Apicomplexa harbor a relict non-photosynthetic plastid, the apicoplast, acquired by the secondary endosymbiosis of a red alga (Janouskovec et al., 2010). The apicoplast lost photosynthetic capability during the conversion to a parasitic lifestyle (Botte ´et al., 2013). However, it still contains plant-like pathways, including a prokaryotic type II fatty acid syn-thesis pathway (FASII) (Waller et al., 1998). The apicoplast is essential for parasite survival in both T. gondii and P. falciparum (MacRae et al., 2012). However, the FASII pathway is thought to be essential only during specific life stages. Indeed, in Plasmodium, disruption of FASII was demonstrated to be dispensable in asexual blood stages but essential for late liver stage in rodent malaria parasites and for sporozoite schizogony during mosquito stages (Vaughan et al., 2009). Nevertheless, changes in P. falciparum blood stage growth conditions, such as lipid starvation during in vitro growth and physiological stress in human patients, induced re-activation of apicoplast FASII [START_REF] Daily | Distinct physiological states of Plasmodium falciparum in malaria-infected patients[END_REF]Botte ét al., 2013), suggesting plasticity of FASII in response to nutritional environment. In T. gondii, FASII is essential during tachyzoite development (Mazumdar et al., 2006).

Apicomplexan parasite membranes are constituted of up to 80% phospholipid (PL), primarily phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI; Welti et al., 2007;[START_REF] Gulati | Profiling the Essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum[END_REF]. T. gondii can readily scavenge PL and triacylglycerols (TAGs) from the host but is also capable of, and dependent on, de novo synthesis of several PL classes (Hu et al., 2017;Amiar et al., 2016;Nolan et al., 2017). Like other eukaryotes, apicomplexan de novo PL synthesis is initiated by the assembly of fatty acid (FA) (i.e., esterification onto a glycerol-phosphate backbone) into specific PL precursors. In T. gondii, FAs to be used for PLs synthesis derive from three sources: (1) apicoplast FASII generating short FA chains (C12:0, C14:0, and C16:0) (Ramakrishnan et al., 2012), ( 2) FA elongases located on the parasite endoplasmic reticulum (ER) generating C16:1, C18:1, C20:1, C22:1, and C26:1 (Dubois et al., 2018), and ( 3) FAs directly scavenged from the host cell [START_REF] Bisanz | Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from apicoplast-generated fatty acids versus scavenging of host cell precursors[END_REF]. Lipidomics reveals that most T. gondii PLs are hybrid/patchwork molecules, comprising one FA moiety from the apicoplast de novo synthesis pathway and a second one scavenged from the host (Amiar et al., 2016). Thus, both scavenging and de novo synthesis of FA are critical for intracellular development.

Typically, phosphatidic acid (PA) is the central precursor for the de novo synthesis of all PL classes by the two-step esterification of FAs onto a glycerol-3-phosphate backbone; first by glycerol-3-phosphate acyltransferases (GPATs) to form lysophosphatidic acid (LPA) and then by acyl-glycerol-3-phosphate acyltransferases (AGPATs) to convert LPA to PA. In eukaryotic cells, GPATs and AGPATs of diverse origins work as a set at several locations within the cell. Apicomplexans have two sets of acyltransferases: one plastid-like set putatively in the apicoplast (prokaryotic pathway) (Amiar et al., 2016) and another pair predicted to be in the ER (the so-called eukaryotic pathway). In T. gondii, the apicoplast GPAT, TgATS1 is essential for tachyzoite development, where it generates LPA from apicoplast-FA for the bulk synthesis of PL (Amiar et al., 2016). The role of parasite AGPATs for lipid synthesis is yet to be determined. Beyond their roles as lipid precursors, PA (and LPA) also have important biophysical properties by controlling the formation of positive or negative membrane curvatures, and thereby influence the recruitment of proteins involved in membrane fusion/fission events such as endocytosis in other eukaryotic models [START_REF] Schmidt | Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid[END_REF][START_REF] Kooijman | Spontaneous curvature of phosphatidic acid and lysophosphatidic acid[END_REF][START_REF] Brown | The lysophospholipid acyltransferase antagonist CI-976 inhibits a late step in COPII vesicle budding[END_REF].

Here we characterize T. gondii AGPATs, focusing on one localized to the apicoplast, TgATS2. We confirm that TgATS2 is an acyltransferase by heterologous complemention of a bacterial mutant. We then generate a knockout (KO) mutant, which was defective parasite cytokinesis and normal lipid profile. Particularly, the impact of LPA/PA changes on the localization of a dynamin-related protein (Drp), TgDrpC, in the TgATS2 mutant is described and provides a rationale for cytokinesis defects associated with drug inhibition of apicoplast FASII [START_REF] Martins-Duarte | Ciprofloxacin derivatives affect parasite cell division and increase the survival of mice infected with Toxoplasma gondii[END_REF]. Finally, changes in parasite lipid composition and lipid fluxes led us to subject parasites to lipid starvation to explore how host nutritional environment affects parasite growth. Analysis of lipid fluxes and growth screening under adverse lipid conditions show that parasites can sense the environment and respond by ( 1) upregulation of de novo lipid synthesis in the apicoplast and ( 2) manipulation of the human host through vesiculation from host organelles and import of such material to the parasitophorous vacuole (PVM) mediated by export of parasite effectors to improve their lipid scavenging. Our analysis provides unprecedented mechanistic insights into parasite metabolic adaption under host nutritional challenge, which was poorly understood until now.

RESULTS

Deletion of Toxoplasma gondii Apicoplast Acyltransferase TgATS2 Results in Aberrant Cytokinesis and Residual Body Formation during Replication

To explore de novo PA synthesis in T. gondii, we searched the genome for AGPATs capable of catalyzing the esterification of an activated FA (i.e., acyl-CoA or acyl-ACP) onto LPA to make PA. We found two AGPAT candidates with conserved motifs: ToxoDB: TGME49_297640 and ToxoDB: TGME49_240860. Phylogenetic analyses reveal that TGME49_297640 clusters with the prokaryotic clade of the pathway with plant and algal sequences and that TGME49_240860 clusters with the eukaryotic clade of the pathway (Figure S1). We termed these enzymes TgATS2 and TgAGPAT on the basis of the plant and eukaryotic terminology, respectively. We generated parasite lines expressing TgATS2 and TgAGPAT endogenously tagged at the C terminus with a triple HA tag, under control of their respective promoters (Figures S2A-S2C). Immunofluorescence assays (IFAs) confirmed TgATS2 targets to the apicoplast (Figure 1A), and transient expression of TgAGPAT-HA showed a perinuclear structure corresponding to the parasite ER (Figure 1B).

To test if TgATS2 and TgAGPAT are functional AGPAT, we complemented an E. coli temperature-sensitive mutant SM2-1 DplsC lacking AGPAT activity [START_REF] Coleman | Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity[END_REF] with recombinant TgATS2 and TgAGPAT. All transformants grew at the permissive temperature of 30 C (Figure 1C). Only those complemented with bacterial EcPlsC and TgATS2 grew at the non-permissive 42 C (Figure 1C). Constructs with TgAGPAT including or not its long N-Ter extension did not grow at 42 C, likely because of TgAGPAT eukaryotic origin (Figure S1). Indeed, eukaryotic AGPATs favor acyl-CoA substrates over acyl-ACP substrates used in bacterial and plastid systems. Thus, it is unclear if TgAGPAT has acyltransferase activity, but TgATS2 complements defective E. coli SM2-1 AGPAT enzymatic activity in vivo, confirming LPA-to-PA synthetic capability.

To investigate the importance of apicoplast TgATS2 during tachyzoite life stages, the TgATS2 locus was disrupted to generate knock-in (KI) and KO mutants of TgATS2 using CRISPR-Cas9 strategies (Figures S2D-S2F). Loss of the protein product was confirmed by western blot (Figures 1D and1E), IFA (Figure 1F), and PCR (Figures S2E andS2F). Both DTgATS2 mutants were viable, but plaque and replication assays revealed that DTgATS2 had a mild yet significant growth defect with significantly more small (two to four parasites) vacuoles and significantly fewer large vacuoles (Figures 1G-1I).

Parasite egress was significantly affected in the DTgATS2 mutant (Figure S2G), but invasion ability showed no difference with the parental line, nor was there a defect in microneme secretion (Figures S2H andS2I). Morphology of different intracellular tachyzoite organelles showed no obvious defects except the apicoplast displaying a mild biogenesis defect (Figures S2J andS2K).

Upon closer inspection of parasite morphology, DTgATS2 parasites appeared fused to each other at their basal poles (Figure 2A), suggesting a cytokinesis defect, which provided a rationale for the egress defect (Figure S2G). Cytokinesis was monitored by localizing MORN1, which curiously displayed no obvious basal mis-localization (Figure 2B). However, DTgATS2 parasites showed important enlargement of the residual body (Figure 2C). Residual body size was quantified by IFA using GAP45 antibody as a marker for the inner membrane complex (IMC). DTgATS2 parasites displayed a significantly larger residual body at the center of big vacuoles (more than four parasites), (Figures 2B and2C). Accordingly, egressed extracellular parasites often remained tethered at their basal pole via a plasma membrane (PM) structure (Figure 2D). Electron microscopy (EM) of DTgATS2 parasites supported the segregation defects seen by IFA (Figure 2E). In dividing parasites, it is commonly seen that the PM is kept connected between two recently divided cells so that the cells are stuck together and distributed to daughter cells during cytokinesis. Very little is known on the molecular mechanisms of PM segregation during cytokinesis. In the parental parasites, initial steps of endodyogeny showed the formation of the daughter cell apical pole along with organelle division before the formation of the daughter cells within the mother cell (Figure 2E). Emergence of the daughter cells initiates the apical-to-basal biogenesis of their PM, partly recycled from the mother (Figures 2E1-2E5), and ends by a constriction of both IMC and PM at the basal poles, leaving a small basal residual body (Figure 2E6). In contrast, there were many division and cytokinesis defects in DTgATS2, which were unable to separate, although a new round of daughter formation could be initiated (Figure 2E1). Furthermore, parasite organelles were frequently found in the residual body as if ejected from improper segregation, likely contributing to the enlarged residual body phenotype (Figure 2E). Affected vacuoles thus displayed enlarged residual bodies that often contained various organelles-including the nucleus, mitochondrion, acidocalcisome vesicles, and other cytosolic material-that appeared to be ejected from the dividing cells because of improper segregation (Figures 2E3 and2E4).

Pieces of mitochondria were a particular feature within enlarged residual bodies (Figures 2E5 and2E6).

We attempted to disrupt TgAGPAT using CRISPR-Cas9 as per DTgATS2 in wild-type (WT) (DTgAGPAT) and DTgATS2 (DTgAGPAT-DTgATS2) genetic backgrounds, but parasites were not viable (Figures S2L-S2O), suggesting that TgAGPAT is indispensable, consistent with its phenotype score (Sidik et al., 2016).

TgATS2 Disruption Reduces C14:0 FA Incorporation into T. gondii Lipids, Skews the LPA/PA Ratio, and Alters PL Abundance and Composition To investigate the role of TgATS2 in lipid metabolism, we performed lipidomic analysis on the DTgATS2 mutant. Disruption of TgATS2 resulted in a large significant reduction of the relative amount of C14:0, the main product of the apicoplast FASII (Figures 3A and 3B). Significant decreases in C18:1 and C20:1 were also observed (Figures 3A and3B). In contrast, there were significant increases in the abundance of C18:0, C22:1, C24:1, C20:4, C20:5, and C22:6 (Figures 3A-3C), most of which can be scavenged from the host, such as C20:4 and C20:5 (Welti et al., 2007;Ramakrishnan et al., 2012;Amiar et al., 2016;Figures S3A and S3B). Comparison of the relative FA abundance between DTgATS2 and its parental line showed significant decreases of C14:0, C20:0, C20:1, and C22:2 (Figure 3C). These results indicate that in addition to the aforementioned cytokinesis defect, DTgATS2 has a highly modified lipid content that relies more on long-chain FAs scavenged from the host (Figures 3A-3C).

To further investigate DTgATS2 lipid defects, we analyzed and quantified each PL class and its individual FA content. The DTgATS2 mutant accumulates significantly more LPA compared with the control parental line and significantly less PA (Figures 3D and3E), consistent given that LPA and PA are the likely substrate and product, respectively, of ATS2 (Figure 1C). The slight reduction in PA suggests that TgATS2 is not responsible for the bulk PA synthesis but rather for a specialist function. Importantly, the LPA/PA ratio was significantly affected in DTgATS2 (Figure 3E). We investigated diacylglycerol (DAG) and other related PLs, namely PC, PE, PI, PS, phosphatidylglycerol (PG) and cardiolipin (CL; Figure 3D). The relative abundance of both DAG and PG significantly decreased in DTgATS2 (Figure 3D). This is relevant because DAG can be a direct product of PA, and PG is the sole PL made from PA in plant chloroplasts [START_REF] Ohlrogge | Lipid biosynthesis[END_REF]. In contrast, the relative abundance of PS, PI, and PE increased in the mutant (Figure 3D).

We then examined the FA profiles of each of these lipid classes. LPA had significant increases in the amounts of C16:0 and C18:0 in DTgATS2 parasites, whereas significant decreases in the apicoplast-specific FAs C12:0 and C14:0 were measured in the mutant (Figure 3F). No major difference was observed in PA composition in DTgATS2 parasites (Figure 3G). Strikingly, though, DAG, PC, PI, and PE all had significantly reduced C14:0 content (Figures 3D andS3C-S3H), which is the main product of FASII and is used by TgATS1 for bulk de novo synthesis of PC, PI, and PE (Amiar et al., 2016). This indicates that TgATS2 likely uses apicoplast-generated C14:0 as its major substrate to make these lipids. In contrast, the levels of two long polyunsaturated FAs (PUFAs), C20:4 and C20:5, in all three major PLs (PC, PI, and PE) were significantly increased in DTgATS2 (Figures S3C-S3H), which is again consistent with mutant parasites compensating for the lack of de novo-made FAs by increasing scavenging long-chain FAs from the host. The FA composition of fetal bovine serum (FBS) and HFF host cells (Figures S3C-S3H) confirmed that C20:4 and C20:5 PUFAs were (F) Electron microscopic image of DTgATS2 mutants reveals important cytokinesis defects: major enlargement of the PM, defects in mother cell membrane constriction and cell daughter attachment at the basal pole (F1 and F2; enlarged in F1 0 , F1 00 , and F2 0 , white arrows), and IMC fragmentation at the separation sites between dividing parasites (F2 00 ). Residual bodies containing unevenly separated nuclei (F1, F3, F3 0 , F4, and F5), mitochondria (F4, F5, F6, and F6 0 ), and acidocalcisomes (F5). N, nucleus; Mt, mitochondria; rb, residual body; Ac, acidocalcisome. Scale bars, 1 mm. present from the host environment. This is consistent with the DTgATS2 mutants' increasing scavenging of these PUFAs to make PC, PI, and PE. We complemented DTgATS2 and WT parasites (Figure S3I) using exogenous PA(14:0;14:0), the putative product of TgATS2, and PA(16:0;18:1) as host-derived PAs. Proliferation assays showed that both exogenous PA sources could significantly boost parasite growth (Figures S3I andS3J) but could not rescue DTgATS2 growth phenotype. This indicates that the PA source needs to be made de novo via TgATS2 for proper division. Because parasites are capable of scavenging lipids from the host and medium, we determined whether the DTgATS2 imported more PA, using PC as a control. DTgATS2 imported significantly more PA and PC than the parental control line (Figure S3K). Together these data on extracellular DTgATS2 corroborate our lipidomic analyses (Figure 3), indicating that the mutant scavenges more lipids to compensate for reduced de novo synthesis.

Disruption of TgATS2 Induces a Mis-localization of the Parasite DrpC Perturbing Parasite Cytokinesis, IMC Formation, and PM Stability Lipidomic analyses revealed a drastic LPA/PA imbalance in the DTgATS2 mutant (Figure 3E). LPA and PA have important structural influences on membrane architecture and endocytosis by inducing local membrane curvatures, which can affect the recruitment and functions of specific dynamins at precise membrane domains for organelle/vesicle fission [START_REF] Adachi | Coincident phosphatidic acid interaction restrains Drp1 in mitochondrial division[END_REF][START_REF] Schmidt | Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid[END_REF][START_REF] Gras | An endocytic-secretory cycle participates in Toxoplasma gondii in motility[END_REF]. For example, synaptic vesicle transport between neurons requires a protein complex composed of a dynamin and an endophilin that exert acyltransferase activity to create the proper membrane groove where the dynamin can pinch and release the synaptic vesicle, or in human mitochondrial fission by the protein Dynamin-like 1, HsDrp1, which requires insertion, recruitment, and regulation through PA. In T. gondii, there are three known dynamin-related (legend continued on next page) Cell Reports 30, 3778-3792, March 17, 2020 3783 proteins (Drps): TgDrpA, TgDrpB, and TgDrpC. TgDrpA and TgDrpB have roles in apicoplast fission and secretory organelle biogenesis, respectively (van Dooren et al., 2009;[START_REF] Breinich | A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii[END_REF]. TgDrpC was recently localized to the basal poles of dividing daughter cells [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF]. We generated a parasite line expressing TgDrpC fused to a 3xHA tag under the control of its endogenous promoter using CRISPR-Cas9 (Figures S4A andS4B) and localized TgDrpC-HA during the tachyzoite intracellular division cycle in DTgATS2 and its parental line (Figure 4A). In parental-line parasites, TgDrpC-HA clustered in small punctate-like compartments in the apical post-Golgi area during interphase (Figure 4A). During daughter budding, TgDrpC re-localized to form two distinct ring-like structures coinciding with the growing ends of the IMC from the budding daughter cells, which constricted at the base of the mother cell during cytokinesis and eventually formed basal caps on the each newly divided parasite (Figures 4A and4B).

In DTgATS2, localization of TgDrpC-HA was only mildly affected during interphase but was drastically affected during division (Figures 4A and4B). Indeed, TgDrpC-HA frequently failed to form the typical ring structures at daughter cells (Figures 4A and 4B). Instead, DrpC-HA was scattered in the cytosol, or formed rings pushing on the side of mother IMC, or improperly constricted at the daughter basal pole (Figures 4A and4B). This contrasted with the normal localization of MORN1, which appears to be a more cytoskeletal component, as its localization remains unaffected during endodyogeny (Figure 2B). We further examined other known interactors of DrpC and thus localized the dynamin-like protein EPS15 [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF] by C-terminally tagging by CRISPR-Cas9 (Figures 4C and4D). In parental strains, EPS15-HA localized to clear punctate dots during interphase similarly to DrpC. During endodyogeny, EPS15 remained as punctate dots and did not re-localize to the daughter rings like DrpC (Figure 4C). In DTgATS2 background, EPS15 was unaffected during interphase though more scattered than in the parental line. However, during endodyogeny, EPS15-HA mis-localized in the cytosol of the parasite when expressed in the DTgATS2 background (Figure 4C), consistent with its role as a DrpC interactor [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF].

In silico sequence alignment showed that (1) TgDrpC is the closest TgDrp homolog to the HsDrp1, which allows mitochondrial fission through its interaction with PA via its Stalk domain including a loop with specific hydrophobic residues [START_REF] Adachi | Coincident phosphatidic acid interaction restrains Drp1 in mitochondrial division[END_REF][START_REF] Adachi | An unstructured loop that is critical for interactions of the stalk domain of Drp1 with saturated phosphatidic acid[END_REF], and (2) the Stalk domain and the PA binding loop seem conserved in TgDrC (3) but are absent in TgDrpA and TgDrpB (Figures S4C-S4E). To confirm this, we tagged and monitored the localization of other TgDrps in the DTgATS2 background. No obvious change in localization of TgDrpA was observed in DTgATS2 parasites, even during the fission of the apicoplast (Figure S4B).

On the basis of homology with HsDrp1, we disrupted the putative PA-binding region of TgDrpC. We expressed this TgDrpC-DPA version of the protein in the parasite to test the importance of the putative PA-binding domain for the localization of TgDrpC. To do so, we transfected a WT TgDrpC-HA cell line with a Cas9-RFP and a PCR product targeting the DrpC PA domain. IFAs on parasites with no Cas9-RFP had typical DrpC-HA localization (Figure 4D). However, parasites with positive Cas9-RFP expression showed that DrpC was mis-localized and scattered throughout the cytosol in a similar manner as in TgDATS2 parasites. These results are also consistent with the cytosolic mis-localization of truncated TgDrpC, excluding the putative PA-binding domain recently reported [START_REF] Melatti | A unique dynamin-related protein is essential for mitochondrial fission in Toxoplasma gondii[END_REF].

Further detailed evidence of improper cytokinesis could be observed under EM. In the parental line, initial steps of endodyogeny showed the formation of the daughter cell apical pole along with organelle division before the formation of the daughter cells within the mother cell (Figure 4E). Emergence of the daughter cells initiates the apical-to-basal biogenesis of their PM, partly recycled from the mother (Figures 4E1-4E5), and ends by a constriction of both IMC and PM at the basal poles, leaving a small basal residual body (Figure 3F6). IMC biogenesis and aberrant endocytosis can be seen in TgDATS2 cells upon closer inspection under EM. In contrast DTgATS2 were unable to separate, although a new round of daughter formation could be initiated (Figure 4F1). Daughter cells were found tightly apposed at normal emergence sites, and their PMs were often missing between daughter IMCs. Instead, interconnection of PM, vesicles, or cisternae could be observed at these apposition sites and at the basal end of dividing cells (Figures 4F1 and4F2). Mother cells were frequently observed to be fused to each other, with vesicle fusion frequently occurring between the two at the site of the PM (Figure 4F2). These defects suggested issues at the PM composition and/or problems in membrane fusion/fission sites. Furthermore, there was no constriction of both IMC and PM from daughter cells, resulting in enlarged residual bodies containing organelles and cytosol portions (Figures 4F3 and4F4). In particular, these membrane invaginations were frequently seen at the junction between two parasites in a process resembling endocytosis.

(E and F) Electron microscopic observation of endodyogenic division in parental line (E) and DTgATS2 (F). (E1-E3) Endodyogeny starts with the formation of daughter cells (dc) by growth of IMC (white arrows) and organelle segregation. IMC scaffolding then grows toward the basal pole (white arrows) encompassing divided organelles (e.g., nucleus N). (E4-E5 0 ) Recycling and biogenesis of PM (black arrow) ends daughter cell emergence from mother cell (mc). (E6 and E6 0 ) Division ends by cytokinesis through constriction of both IMC and PM at basal pole (black arrows) to form a small residual body (rb). (F) DTgATS2 shows an incomplete separation of daughter cells during cytokinesis with absence of PM biogenesis between closely apposed IMC (F1 and F3 and insets, white arrows), presence of vesicle/cisternae inside membrane structures at the inter-IMC space (F2 0 , white arrows), absence of mother IMC (F3 0 , black arrow), absence of basal constriction forming large residual bodies leaving floating daughter IMC (F3, F4, and 4 0 , white arrows). Scale bar, 1 mm. (G and H) Proposed molecular model for TgDrpC function during endodyogeny and cytokinesis in WT parasite (G) and DTgATS2 (H). LPA and PA molecules induce positive and negative curvature, creating grooves in membranes for TgDrpC to insert at specific sites during division for a pinching function during endodyogeny (G).

Nutrient Starvation Enhances the Synthesis of FA by

Apicoplast FASII in T. gondii and Blocks Intracellular Proliferation of P. falciparum Blood Stages Lacking a Functional FASII Because TgATS2 has a role in maintaining parasite lipid homeostasis, we set out to determine the balance of de novo synthesized versus scavenged lipids in DTgATS2 using a stable isotope precursor of apicoplast synthesized FAs, U-13 C-glucose. (Ramakrishnan et al., 2012;Amiar et al., 2016;Dubois et al., 2018). Incorporation of 13 C within FA is detected by increase of mass and determined in relation to non-labeled FA. Distribution of 13 C incorporation to each FA isotopologue is shown as its own mass (M) plus number of 13 C carbon incorporation (i.e., M + x). In both parental and DTgATS2 mutant lines, we observed significant differences of 13 C incorporation in C14:0, C16:1, C18:0, and C18:1 (Figure 5A). Isotopologue distribution of apicoplastsignature C14:0 showed that DTgATS2 had 13 C incorporation up to M + 14, but major incorporation occurred at lower masses (M + 8, M + 10) than the parental (M + 12, M + 14; Figure 5B). This indicates that FASII is active in DTgATS2 but slowed down in the process of making C14:0, thus explaining the C14:0 reduction previously detected (Figure 3A). Similar significant results were observed for C16:0 isotopologue distribution, although overall incorporation was similar between parental and DTgATS2 (Figure 5C). C18:0 in DTgATS2 had higher 13 C incorporation than the parental, and its isotopologue distribution showed more short FA from the apicoplast (Figure 5D).

Lipidomic analyses thus indicate that both scavenged and de novo lipid fluxes are modified in DTgATS2. To tease out the impact of host nutritional environment on both pathways, we sought to measure parasite lipid fluxes under adverse host nutritional/lipid conditions, through limitations in FBS concentrations in parasite culture media. Interestingly, gas chromatographymass spectrometry (GC-MS) analysis revealed that 13 C incorporation into all FASII-generated and further ER-elongated FA products (i.e., C14:0, C16:0, C16:1, C18:0, C18:1, and C20:1) was significantly higher by 5%-15% under FBS starvation in the parental line (Figures 5E, 5F, andS5A). In addition, 13 C incorporation into most FAs is increased in the WT parental line (Figure 5F). These results suggest that apicoplast de novo FA/lipid synthesis can be upregulated during FBS starvation to compensate for the lack of nutrients in the external environment. However, in DTgATS2, the 13 C incorporation into each FA was decreased by FBS starvation (Figure 5F). No morphological changes could be observed by IFA in the FBS-starved WT or DTgATS2 mutant (Figure S5B). Both parental line and DTgATS2 mutant showed a significant reduction in the synthesis of C18:0 in FBS-starved conditions, suggesting that C18:0 is obtained predominantly by scavenging from the host cell (Figure 5F). Because the availability of lipids from the environment is limited, the FA abundance in the parental line was decreased (Figure 5G). Interestingly however, the FA abundance in DTgATS2 was increased in most of its FA species during FBS starvation (Figure 5G).

Although we observed a defect in the activation of FASII in DTgATS2, FASII was nevertheless viable during FBS starvation. This suggests that if FASII is active, regardless of the level of FASII activity, the parasites are viable under FBS starvation, consistent with its essential role in tachyzoites. However, in P. falciparum, FASII is not essential during nutrient-replete blood stage but is activated under lipid starvation, apparently to compensate for reduced availability of scavenge-able lipids (Yu et al., 2008;Botte ´et al., 2013). Our results in T. gondii led us to re-think the current hypothesis regarding the dispensability of the apicoplast FASII in P. falciparum blood stages and to test the essentiality of malaria parasite apicoplast FASII under nutrient/lipid-starved conditions. We grew P. falciparum FASII KO, DPfFabI (Yu et al., 2008), and its parental line (NF54) in either regular (i.e., lipid-rich) culture medium or in ''lipid-starved'' minimal medium [START_REF] Mi-Ichi | Oleic acid is indispensable for intraerythrocytic proliferation of Plasmodium falciparum[END_REF]Botte ´et al., 2013). Both NF54 and DPfFabI grew normally in the regular culture medium (Figure 5H). In the lipid-starved medium, NF54 was viable but grew significantly slower than in lipid-replete conditions, as previously reported (Shears et al., 2017). However, DPfFabI grew only for the first 2 days in lipid-starved media, but after 4 days, a sharp decrease in growth occurred, and this led to a complete loss of detectable parasites after 8 days and showed no sign of further recovery in the next monitored cycles (Figure 5H). This shows that FASII is required for the malaria parasite blood stages to adapt its lipid metabolism in response to an adverse host lipid environment, a similar situation to that revealed here for T. gondii.

Because environmental FBS starvation induces an increase of de novo lipid synthesis, we investigated the effect the lipidnutrient-depleted conditions (i.e., 0%, 1%, and 10% of FBS) on various mutants involved in lipid metabolism in T. gondii. We assessed parasite growth by plaque assay and quantified plaque area. The WT and parental parasite lines could grow equally well in DMEM supplemented with 0%, 1%, or 10% FBS (Figures 5I-5R and S5C), without affecting the integrity of HFF host cells. FBS starvation reduced growth of DTgATS2 under 0% FBS (Figure 5I). TgATS1-depleted cells grew sharply less in the regular culture conditions (i.e., 1% FBS), but starvation under 0% FBS led to the quasi-absence of plaques, whereas an increase to 10% FBS partially rescued the growth defect seen in 1% FBS (Figure 5J). This suggested that in the absence of the major de novo PL precursor synthesis pathway, the parasite could partially compensate the growth defect by accessing more host lipid resources. The acetyl-CoA synthetase TgACS (Dubois et al., 2018) was adequately responsive to FBS starvation (Figure 5K). Interestingly, proteins not involved in bulk membrane/lipid synthesis, such as TgPKA-iKO, could not be rescued by excess nutrients (Figure 5L; [START_REF] Uboldi | Protein kinase A negatively regulates Ca 2+ signalling in Toxoplasma gondii[END_REF].

Because host FA binding proteins (FABPs) are upregulated upon tachyzoite invasion (Hu et al., 2017), we searched the genome of T. gondii for homologs of FABPs that could be responsible for the transport of FAs in the parasite during starvation but found none. Instead, we found two proteins belonging to the closely related family of acyl-CoA binding protein (ACBP): TgACBP1 and TgACBP2. We found that TgACBP1 and TgACBP2 localized at the parasite cytosol and mitochondrion, respectively (Figures S5D-S5G). We generated inducible knockdown parasite lines for both (Figures S5D andS5E). However, plaque assays showed that both proteins were dispensable during tachyzoite life stages, and neither was responding to FBS starvation (Figures 5M and5N), suggesting that neither of the TgACBPs is involved as an effector for the adaptation to nutritional environment. We generated a TgACBP1 and TgABCP2 double KO and a double ACBP1iKD/sterol carrier protein (SCP2) KO cell line, which we also found to be viable and not responsive to starvation (Figures S5H andS5I).

We then hypothesized that parasite effectors putatively exported into the PVM or toward the host cell could be used by the parasite to collect putative host membrane material generated during FBS starvation. To test this, we investigated TgASP5, a Golgi-resident aspartyl protease that controls the non-canonical trafficking pathway of parasite effectors toward the PVM and the host cell, during FBS starvation [START_REF] Bougdour | Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression[END_REF]. Strikingly, FBS starvation significantly exacerbated the growth defect in DTgASP5 (Figures 5O andS5). By contrast, (A-G) U-13 C-glucose labeling for 72 h to monitor apicoplast FA synthesis by 13 C incorporation to fatty acids (blue, parental line; red, DTgATS2). (A) 13 C incorporation to each fatty acid in 1% FBS. (B-D) Mass isotopologue distribution in 1% FBS for C14:0 (B), C16:0 (C), and C18:0 (D). The x axis shown as ''M + X'' represents mass with ''X'' 13 C atoms incorporated during the FA synthesis. (E) 13 C incorporation to each fatty acid in 0.2% FBS. FASII metabolic activity increased upon FBS starvation in the parental line but not in DTgATS2. (F) Change in 13 C incorporation between 0.2% FBS and 1% FBS (ÀFBS/+FBS). (G) The relative abundance of each FA (ÀFBS/+FBS). (H) Asexual blood stage growth assay of P. falciparum FabI-KO and its parental line (NF54) in regular (lipid-rich) culture medium and lipid-starved medium reveals that FASII is essential in blood stage in low-lipid environment. (I-T) Growth assays conducted in 0%, 1%, or 10% FBS in different T. gondii mutants and strains: TgATS2 (I), TgATS1 (J), TgACS (K), TgPKA (L), TgACBP1 (M), TgACBP2 (N), TgASP5 (O), TgMyr1 (P), TgGRA16 (Q), TgARO1 (R), type II PRU (S), and type II ME49 (T). n R 3. ns, not significant; *p % 0.05, **p % 0.01, ***p % 0.001, and ****p % 0.0001. the mutant cell line DTgMYR1 (the canonical system to export effectors toward the host; [START_REF] Franco | A novel secreted protein, MYR1, is central to Toxoplasma's manipulation of host cells[END_REF] showed overall less growth than the parental cell line, although DTgMYR1 grew equally well among the 0%, 1%, and 10% FBS conditions (Figure 5P). To examine the effects of some specific GRA effectors, we examined a GRA16-KO cell line, which we observed to have a minor but significant growth defect under FBS starvation, suggesting that at least some GRA proteins are important, likely in combination (Figure 5O). We also examined a mutant for rhoptry secretion TgARO-iKO [START_REF] Mueller | The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion[END_REF] but found that under ATc treatment, the mutant died regardless of FBS concentration (Figure 5R), suggesting a primary role in host invasion prior to host re-wiring.

Last, we explored strain-specific differences between in Toxoplasma between the hypervirulent type I RH strain and type II strains (Prugniaud, ME49) capable of forming chronic stages (bradyzoites). Both type II strains showed significantly reduced growth in lipid-depleted medium (Figures 5S and5T), unlike type I strain.

Together, these data provide evidence that in response to nutrient starvation, parasite effectors can be trafficked to the host cell, primarily via the TgASP5 export pathway, likely to enhance the ability to scavenge resources.

Nutrient Starvation Induces the Formation of Multimembrane-Bound Vesicles in Host Cells that Are Taken up by the Parasite To investigate potential changes to the host cell and hence hostparasite interactions during lipid starvation, we performed EM on starved (0%, 1%, or 10% FBS) HFF host cells infected with either the parental parasite line or DTgATS2. Growth in 10% FBS led to no obvious phenotype changes in the hosts cells or the parental parasite line or the DTgATS2 mutant (Figures 6A and6B), but reduction to 1% and 0% FBS induced striking changes in the host cells, which became extensively vesiculated irrespective of whether they were infected with the parental line or DTgATS2 (Figures 6A and6B). Such vesiculation was not observed in uninfected HFF host cells put under nutrient starvation. Giant multi-vesicular bodies (gMVBs; i.e., large membranebound compartments containing various smaller vesicles) were frequent in 1% FBS-grown cells (Figures 6A and6B) and very numerous at 0% FBS (Figures 6A and6B). The gMVBs are distinct from host autophagosome, as they lack the typical double/multiple surrounding membranes and the cytosolic material defining autophagosomes [START_REF] Yla ¨-Anttila | Monitoring autophagy by electron microscopy in mammalian cells[END_REF]. This was confirmed by IFA using the typical autophagosome marker anti-LC3, which showed no accumulation of autophagosome under 10%, 1%, or 0% FBS (Figure S6). The gMVBs could arise from the host ER, as the ER could be seen swelling and forming networks containing large lipid bodies (Figure 6B3). gMVBs were also often seen in close apposition or contact with the mitochondria and/or ER network, indicating that material could also be transferred from both (Figures 6B3 and6B5). However, gMVBs were more often observed arising directly from the host nuclear envelope, potentially a major contributor to their formation (Fig- ures 6B6-6B8). The gMVB accumulated in close vicinity with the PVM, which houses the parasite during its intracellular development and serves as the exchange interphase between the host and the parasite. The gMVBs were not only close to the PVM but appeared to be interacting with the PVM with host material and vesicles from the gMVB, apparently ''percolating'' through the PVM (Figure 6B4) or directly from their originating organelles (Figure 6B5) to eventually be found in the PVM (Figures 6A and6B1). These vesicles appeared in both WT and DTgATS2, suggesting that the host cell is responding to the nutrient deficiency in the same way. The DTgATS2 parasite cytokinesis phenotype (e.g., Figure 2A) was still observed and apparently exacerbated in 0% and 1% FBS growth medium (Figure 6B). This vesicle/gMVB formation and trafficking to and within the PVM was not apparent in high (10%) FBS medium, suggesting that host gMVBs somehow allow the parasite to increase its lipid scavenging in the absence of nutrient rich serum. Together this indicates that gMVBs contain multiple vesicles that ( 1) are dependent and induced by nutrient availability and ( 2) originate directly from diverse host organelles. The gMVBs are distinct from lipid droplets of host cell origin used as a lipid source by T. gondii (Nolan et al., 2017;[START_REF] Romano | The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network[END_REF].

However, it is possible that FBS starvation leads to increased host cell lipid droplet import. Nile red staining confirmed that FBS starvation induced a significant increase of the amount of lipid droplets into the parasites and its PVM (Figure 6C). In contrast, low FBS content resulted in a reduced amount of lipid droplets in uninfected host cells, while high FBS content increased their presence in the host cells alone (Figure 6D). This further indicates that increase of import of lipid droplets to the parasite is upregulated by the parasite during FBS starvation.

Because gMVBs also seem to arise from host mitochondria, we used an anti-lyso-bi-PA (LBPA; i.e., a degradation product of mitochondrial CL) antibody [START_REF] Kobayashi | A lipid associated with the antiphospholipid syndrome regulates endosome structure and function[END_REF], a lipid that can also be scavenged by intracellular parasites [START_REF] Romano | The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network[END_REF]; Figure 6E). LBPA was found surrounding the PVM in the host cell, within the PV and the parasite, but its localization and intensity remained unchanged in response to reduced FBS content (Figure 6E). Direct salvage of mitochondrial CL per se might not be the primary upregulated scavenging pathway during lipid starvation.

To determine whether host mitochondrial sequestration could affect parasite adaptation to low host nutrient, we measured the levels of mitochondrial sequestration in type I parasites (prone to host mitochondrial sequestration) and type II parasites (not sequestering host mitochondria) [START_REF] Pernas | Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response[END_REF]. Host mitochondria was monitored with MitoTracker. Both RH type I and type II ME49 parasites showed no major difference in host mitochondrial sequestration after FBS starvation (Figure S6).

DISCUSSION

We have shown that TgATS2 is an apicoplast acyltransferase able to esterify FAs on LPA to generate PA, a precursor for a wide range of parasite lipids. KO of TgATS2 resulted in perturbed lipid fluxes, which affects LPA/PA lipid balance, causing mislocalization of TgDrpC and vesiculation during cytokinesis. Furthermore, changes in lipid profiles of DTgATS2 showed the capacity of WT parasites to exhibit considerable metabolic plasticity at both de novo FA synthesis in the apicoplast and host modification for organelle membrane scavenging, together critical for adaptation to nutrient-limiting conditions in the host (Figure 7; Table S1).

Roles of PA and LPA in Membrane Curvature and Cell Division Membrane PLs have different physical shapes according to the relative sizes between the polar head and the FA tails. Most PLs are cylindrical, while PA is cone shaped and LPA adopts an inverted cone shape; thus their insertion into membrane bilayers facilitates curvature and in-or evagination [START_REF] Kooijman | Spontaneous curvature of phosphatidic acid and lysophosphatidic acid[END_REF].

Furthermore, in human cells, dynamin pinching requires endophilin-1, an ATS2 homolog, as a partner to create LPA/PA curva-tures [START_REF] Burger | Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid[END_REF][START_REF] Shin | Putting the pH into phosphatidic acid signaling[END_REF], improving penetration of a larger part of dynamin into the lipid monolayer [START_REF] Burger | Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid[END_REF][START_REF] Shin | Putting the pH into phosphatidic acid signaling[END_REF], similar to the relationship between TgATS2 and TgDrpC. Our results reveal the previously unrecognized importance of the apicoplast in maintaining internal lipid homeostasis. Furthermore, the functional role of TgATS2 for PA synthesis during division provides a mechanism for the long-standing question of why drugs targeting the apicoplast display a secondary cytokinetic defect [START_REF] Martins-Duarte | Ciprofloxacin derivatives affect parasite cell division and increase the survival of mice infected with Toxoplasma gondii[END_REF].

Our results nicely complement those of a recent study that identified the basal complex as a major site of endocytosis in (A and B) Transmission electron micrographs of intracellular WT tachyzoites (A) and DTgATS2 mutant parasites (B) grown in 0%, 1%, and 10% FBS. Nutrient starvation (i.e., 0% and 1% FBS) induces formation of giant multi-vesicular bodies (gMVBs) in the host cell (hc), containing various vesicles, including lipid bodylike (white stars). In starvation, gMVBs localized in the cytosol (cyt) in contact with the parasitophorous vacuole (pv) (A1, A2, A4, B1, and B2), and their content was imported through and into the PV (A1, black stars; B2 and B6, black arrows); gMVBs were arising from host endoplasmic reticulum (ER; A2), mitochondria (mt; A2 and A3), and mainly swollen nuclear envelope (N; B3-B5). Ten percent FBS did not induce gMVB formation in both parental and DTgATS2. Scale bar, 1 mm. (C) Nutrient starvation induces a significant increase of lipid droplets within the parasite and its PV as measured by IFA using Nile red (Nile red dots were counted for 100 or more parasites; n = 3; ns, not significant; *p % 0.05, **p % 0.01, ***p % 0.001, and ****p % 0.0001). Scale bar, 2 mm. (D) Nutrient starvation induces a decrease of lipid bodies in uninfected HFF host cells as measured by IFA using Nile red. Scale bar, 2 mm. (E) IFA shows that import into parasites of LBPA (anti-LBPA) is not affected by nutrient starvation. Scale bar, 2 mm. (F) Nutrient starvation induces a significant growth defect in T. gondii tachyzoites ME49 type II strain compared with RH type I strain. p values are as mentioned as above.

motile tachyzoites, consistent with the basal complex localization of DrpC (Figure 4; [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF]. Our results show that endocytosis occurs during intracellular stages and that aberrant LPA/PA ratios caused by the loss of ATS2 disrupt this process.

Furthermore, many DrpC-interacting proteins have been identified as part of a larger endocytic protein complex, including EPS15, AP2 adaptins, and, intriguingly, Kelch13 (Heredero- [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF]. Kelch13 is the infamous protein found mutated in artemisinin-resistant malaria spreading throughout Asia (Menard and Dondorp, 2017). Kelch13 therefore likely has a role in endocytosis consistent with DrpC and other interacting partners [START_REF] Heredero-Bermejo | TgDrpC, an atypical dynaminrelated protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division[END_REF]. Intriguingly, it has been shown that FASII activity is often increased in artemisinin-resistant parasites [START_REF] Chen | Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin-induced dormant ring stages of Plasmodium falciparum[END_REF]. Our evidence here demonstrates that the upregulation of FASII produces LPA that modulates cytokinesis and endocytosis processes. Again, this highlights the previously unrecognized importance of the apicoplast in maintaining internal lipid homeostasis in parasites.

Environmental and Nutritional Conditions Drive the Adaptation of the Apicoplast Metabolic Capacities as

Well as the Scavenging Capacities Importantly, Toxoplasma could increase production of FA in the FASII pathway in nutrient/lipid-deprived medium similarly to P. falciparum (Botte ´et al., 2013). Hence, apicomplexan parasites show high metabolic flexibility to obtain FA for the major membrane building blocks required for growth, as pointed out by recent studies exploring Plasmodium survival in nutrientdepleted conditions (Mancio-Silva et al., 2017;[START_REF] ´s | Dietary alterations modulate susceptibility to Plasmodium infection[END_REF]. Importantly, our results demonstrate that P. falciparum lacking a FASII and grown in lipid-deprived conditions was unable to properly proliferate, ultimately dying. This suggests that apicoplast FASII is facultative rather than totally dispensable in malaria parasite blood stage and can be activated during lipid starvation to meet PL needs. This FASII flexibility is consistent with a growing pool of evidence including the upregulation of FASII and the apicoplast acyltransferase PfG3apiGPAt (a homolog of TgATS1) transcripts in starved patients [START_REF] Daily | Distinct physiological states of Plasmodium falciparum in malaria-infected patients[END_REF] and the essentiality of most FASII enzymes, including the central acyl-carrier protein ACP in both T. gondii and P. falciparum (Sidik et al., 2016;[START_REF] Zhang | Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis[END_REF], summarized in Table S1. Therefore, environmental factors could have important consequences in treating patients. Indeed, if patients are under stress, nutrient deprivation, or malnourished conditions, the FASII pathway could become a secondary target of choice to help eradicate the parasites. Altogether these data question whether isopentenyl pyrophosphate (IPP) synthesis is the sole essential function of the apicoplast during Plasmodium blood stage [START_REF] Yeh | Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum[END_REF]. Rather, our data put the parasite back into its physiological context, where nutrient availability and environmental conditions drive the requirement and regulation of a given metabolic pathway. Furthermore, the scavenging of Toxoplasma can also be seen to be upregulated through exported effectors by evidence that ASP5 KO is partially rescued by excess host lipids and the induction of host remodeling to make gMVBs, although the identity of these gMVBs warrants further investigation. This redefines what we call an essential gene, where phenotypes might only be seen under starvation conditions.

A major question raised here is the nature the signaling factor(s) responsible for environmental sensing and metabolic adaption of both apicoplast de novo synthesis and scavenging pathways. Both T. gondii and P. falciparum lack the canonical mTOR-based nutrient-sensing pathways present in other eukaryotes, but a recent study showed that P. berghei is capable

LEAD CONTACT AND MATERIALS AVAILABILITY

Materials generated in this study are available upon request. Information and requests for resources and reagents should be directed to the Lead Contact, Cyrille Botte (cyrille.botte@univ-grenoble-alpes.fr). Plasmids and parasite lines generated in this study will be made freely available by the Lead Contact upon request which may require the completion of a Material Transfer Agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

T. gondii culture Toxoplasma gondii parental lines RH TATi1-DKu80 (Sheiner et al., 2011) and RH-DKu80 (Huynh and Carruthers, 2009) and derived transgenic cell lines were grown in confluent human foreskin fibroblasts (HFFs) in high glucose DMEM supplemented with 1% Foetal Bovine Serum, as described (Amiar et al., 2016). ME49 parental cell cultures were additionally supplemented with 10 mM HEPES.

P. falciparum culture P. falciparum NF54 wild-type parasites were maintained as previously described [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]. Briefly, Plasmodium blood stage parasites were maintained at 2% hematocrit in 1640 RPMI-HEPES supplemented with 10% AlbuMAX II (GIBCO) and 0.25% gentamycin. Parasites were grown sealed Perspex chambers gassed with beta mix gas (1%O 2 5%CO 2 , 94% N 2 ) at 37 C and maintained on 48-hour cycles.

METHOD DETAILS

Gene identification and sequence analysis

Gene identification and sequence analysis

Arabidopsis thaliana sequence of ATS2 (GenBankTM and TAIRTM IDs: NP_194787 and AT4G30580 respectively) was used as a query sequences for BLAST searches against the Toxoplasma gondii genome on ToxoDB database (https://www.toxodb.org/). Phylogenetic analysis of AGPAT related proteins was performed on the Phylogeny.fr platform [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the non-specialist[END_REF]. Protein sequences were then aligned by ClustalW software [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] and the maximum likelihood phylogeny was generated using the PhyML [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]. We generated multiple sequence alignment using Clustal Omega [START_REF] Li | The EMBL-EBI bioinformatics web and programmatic tools framework[END_REF].

T. gondii plasmid constructs

Plasmid LIC-3HA-DHFR was used to generate a 3 0 endogenous tagging with 3xHA coding sequence of ToxoDB: TGME49_297640 (TgATS2) and ToxoDB: TGME49_240860 (TgAGPAT). A 2229 bp fragment corresponding to the 3 0 of TgATS2 was amplified from genomic DNA using primer sets 5 0 -TCCTCCACTTCCAATTTTAGCGTTCGTCTCGGTGGCGGC-3 0 and 5 0 -TACTTCCAATC CAATGCTTCAGACACTCGGTGCAAA-3. A 5466 bp fragment corresponding to promoter and gene sequence of TgAGPAT was amplified using primer sets 5 0 -TACTTCCAATCCAATGCAGCCAGCAAAGGACGAAAGG-3 0 and 5 0 -TCCTCCACTTCCAATTTTAGC GAGACCGTGGCCTCGGTGGG-3 0 . These fragments were cloned into pLIC-3HA-DHFR vector as described previously (Huynh and Carruthers, 2009). Vectors LIC-TgATS2-3HA-DHFR and LIC-TgAGPAT-3HA-DHFR were confirmed by PCR screen using primer sets 5 0 -GCATAATCGGGCACATCATA-3 0 and 5 0 -ATACGCATAATCGGGCACATCATA-3 0 and by sequencing (Eurofin genomicsTM).

e4 Cell Reports 30, 3778-3792.e1-e9, [START_REF] Romano | The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network[END_REF] Plasmid pTOXO_Cas9-CRISPR (gift from Hakimi Lab, Grenoble, France) was used to integrate a gRNA within BsaI restriction site as previously described [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]. Briefly, Crisp-Fwd and Crisp-Rv primer sets were phosphorylated and annealed: TgATS2-KI: 5 0 -AAGTTACGGGTGTGCGCCGCCTTGCG-3 0 and 5 0 -AAAACGCAAGGCGGCGCACACCCGTA-3 0 , TgATS2-KO: 5 0 -AAGTTG GAGCGCCGACGGGCGACTGG-3 0 and 5 0 -AAAACCAGTCGCCCGTCGGCGCTCCA-3 0 , TgAGPAT-KO: 5 0 -AAGTTCTCTGCCGAGT TCCAATCGCG-3 0 and 5 0 -AAAACGCGATTGGAACTCGGCAGAGA-3 0 . The gRNAs were then ligated into pTOXO_Cas9-CRISPR plasmid linearized with BsaI, yielding pTOXO_Cas9-CRISPR::gTgATS2-KI, pTOXO_Cas9-CRISPR::gTgATS2-KO and pTOXO_Cas9-CRISPR::gTgAGPAT-KO, respectively.

For TgATS2 knockout by CRISPR-CAs9, an appropriate HXGPRT cassette amplified by PCR from pMini (kind gift from the Hakimi laboratory) using those primer sets, TgATS2-KI: 5 0 -GAGGCCCTGCGTCTCCTCAAGCG-AAAGGCGCCGCCACAGTCGACGGGTGT GCGCC-GCCTCAGCACGAAACCT-TGCATTCAAACC-3 0 and 5 0 -GCTACTCCTTCTTCCCCTCTCG-CGTTGTGTGTCTCCCCGTCG CGTTCTGCGTCGCCAGCAGTGTCACTGTAGCCTGCCAGAACA-3 0 ; TgATS2-KO: 5 0 -GACACACAACGCGAGAGGGGAAGAAGGA GTAGCTCTCG-TCGCCTTTCCAGAAGGTACTCCAGCACGAAACCTTGCATTCAAACC À3 0 and 5 0 -CTTCG-CTGCTCGTTCGTCTTC ATGTGGGGAAGGAGCAGCACGAAACCTTG-CATTCAAACC-3 0 .

DrpA and DrpC were localized by CRISPR Cas9 strategy. Guides were inserted into Cas9 U6 universal plasmid [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]) by either standard ligation of annealed primers or Q5 mutagenesis. Cells were transfected together with PCR product encoding either HA3-CAT and DrpC homology flanks, for DrpC or GFP sequence without selection and DrpA homology flanks for DrpA.

For DrpC HA3 CAT CRISPR Cas9 tagging, DrpC was tagged at the 3 0 terminus by CRISPR Cas9 [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]. For DrpC, the protospacer gaatggggcttgaaactgtg was chosen and primers 5 0 AAGTTgaatggggcttgaaactgtgG 3 0 and 5 0 AAAACcacagtttcaagccc cattcA 3 0 were annealed together and ligated into U6 universal plasmid [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]. The HA3-CAT cassette was PCR amplified by primers with 50 bp homology flanks (FOR aggaagttccggtcggcttccgtcaccgtt-gaatggggctAAAATTGGAAGTGGAGGACGGG and REV gttcttcccagtgctctggcga-agtgggccagcacaagccaGTTGTAAAACGACGGCCAGTG) and overhang corresponding to the 3 0 end of DrpC and in frame with HAx3. 50 mg of both plasmid and PCR product were transfected and placed under chloramphenicol selection [START_REF] Kim | Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker[END_REF]. DrpC was also localized by pLIC-HA3-CAT using primers TACTTCCAATCCAATTTAGCgcacggtctgttgttc tacg and TCCTCCACTTCCAATTTTAGCagccccattcaacggtg (Sheiner et al., 2011). For DrpA, the protospacer gatggaggagttgattcctg was inserted into the Universal Cas9 Plasmid using the NEB Q5 site directed mutagenesis Kit with the oligos 5 0 gatggaggagtt gattcctgGTTTTAGAGCTAGAAATAGC 3 0 and 5 0 AACTTGACATCCCCATTTAC 3 0 . PCR product was amplified using 5 0 -gttgccctggctt cctcctctttctctcctctctctcaagATGGCGGTGAGCAAGGGC3 0 5 0 -cgtcctgcaggcgattgacAacaggaatcaactcctccatCCCGGGCTTGTA CAGC 3 0 using GFP cDNA as a template and co-transfected with U6 guide RNA plasmid described above. Transfected parasites were seeded onto coverslips and then transiently observed after 24 hours growth.

For the DrpC PA domain mutation, a guide was identified within a DrpC exon and the 20 bp protospacer (5 0 ggcgagctgatcctcgag gt 3 0 ) was inserted into a CRISPR Cas9 plasmid using Q5 with primers 5 0 ggcgagctgatcctcgaggt-GTTTTAGAGCTAGAAATAGCA AG 3 0 and 5 0 AACTTGACATCCCCATTTAC 3 0 [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]. The PCR product was amplified with the following primers 5 0 cgtcgccttgtacggaaacgctt-ggagacgcaaaaacggattGCGGTGAGCAAGGGCG 3 0 and 5 0 agccccattcaacggtgacggaagccgaccggaacttcc tgcCCCGGGCTTGTACAGC 3 0 . The guide and the PCR product were transfected together and parasites were seeded onto HFFs on coverslips and grown for 24 h. Cells were labeled with anti-IMC and anti-HA antibodies and viewed under the microscope. Cas9 expression was visualized by the Cas9-RFP tag to observe parasites with DrpC-HA normally (Cas9-RFP absent) or with the PA binding domain disrupted (Cas9-RFP present).

For TgACBP1i-HA KD, the 5 0 UTR flank was PCR amplified using primers ACACGGGCCCACGATCAGTTGAGTTCCGAGG and GACACATATGAAGG -TCGAAAGAAGGCTCC and inserted into ApaI/NdeI sites of pPR2-HA3 (Katris et al., 2014). The 3 0 flank was amplified using primers CTTGCCCGGGATGGCCTCGCgtaaggaagg and CAGAGCGGCCGCCTGT -GTCGTGAGCGAGTGAC and then inserted in frame with a Tet7O/SAG4 promoter using XmaI/NotI sites. Plasmid was linearized with NotI prior to transfection and selected using pyrimethamine. For ACBP2, flanks were PCR amplified using respective primers below into the plasmid pPR2-GFP or pPR2-mCherry (adapted from pPR2-HA3, (Katris et al., 2014). The 5 0 UTR flank was amplified using CTGAGGGCCCGC GACGCTCCAGAAGACTCC and GTACCATA -TGTTATTATATGGTTGAAAGAAGC inserted first using ApaI/NdeI sites. Next, the 3 0 UTR flank was amplified using GACTGATATCGATT -ACGGCTTCAACTCCGTC and ATTAGCGGCCGCCTTCATAGGAC -CAGAGCC and inserted using MscI/NotI sites. ACBP2 cDNA sequence was amplified using GATCAGATCTAAAATGGCGAGGCCTGTA CATCTTGGG and GTACCCTAGGAGTAGCTTTTGAGGCGGTG inserted last into BglII/AvrII sites then selected using pyrimethamine. ACBP1 pLIC was PCR amplified using pLIC primers TACTTCCAATCCAATTTAGCTACAACGGAGCAGACAGAGG and TCCTCCACTTCCAATTTTAGCCGCGCTTTTCTCGCGCC into pLIC-HA3-CAT (Sheiner et al., 2011), and linearized prior to transfection and selection on chloramphenicol.

For ACBP1 KO/ACBP2iKD the following protospacer was selected, 5 0 GGGGCGTTCCACTGAGAGAA 3 0 , inserted into a U6-Cas9 expression construct [START_REF] Sidik | Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[END_REF]. A PCR product w homology flanks for ACBP1 was made with the following primers 5 0 ATTTTTTCCAAAGTCCATGCTGGGTTTCTCCCCTG-TGTCTAGGGAGCCTTAAAACCCTCGAAGGCTGCTAGTAC 3 0 and 5 0 AGATGATTTGACGACACGCGCCTCGGAAGTCGCTCTGTTTACG-CGCTTTTTGCCAGAACACTTGTCAACCG3 0 using a graCAT-sag-mcherry resistance cassette as a template and transfected with U6 construct and selected for with Chloramphenicol. For SCP2 KO/ACBP1iKD, the following protospacer5 0 GTACGCTTGCTGTGGAAAAA 3 0 was inserted into a U6-Cas9 construct and co-transfected with the following primers 5 0 gaacaggtgctgacacttgctcgagaatcctgtcgctgcaagttctgagttAAAACCCTCGAAGGCTGCTAGTAC 3 0 and 5 0 agggcgagtttcacgaaatc ttcgt -ccaacaaagtgatggtgcagtcgcaTGCCAGAACACTTGTCAACCG 3 0 using a graCAT-sag-mcherry resistance cassette and selected for with chloramphenicol.

T. gondii transfection RH-DKu80 parasite line was transfected with 100 mg of pLIC-TgATS2-3HA-DHFR linearized with BlpI for stable integration of HA-tag at C terminus of TgATS2. 150 mg pTOXO_Cas9-CRISPR::gTgATS2-KO and pTOXO_Cas9-CRISPR::gTgAGPAT-KO were transfected in TgATS2-HA line with 10 mg of appropriate HXGPRT cassette for TgATS2-KI and TgATS2-KO, PCR product as described above. Electroporations were performed in a 2-mm cuvette in a BTX ECM 630 (Harvard Apparatus, at 1,100 V, 25 U, and 25 mF. Stable lines expressing the tagged constructs were selected selected in media with 1 mM pyrimethamine or 25 mg/ml mycophenolic acid and 50 mg/ml xanthine and cloned by limiting dilution.

RH-DKu80 parasites were also transiently transfected with pLIC-TgAGPAT-3HA-DHFR. pTOXO_Cas9-CRISPR::gTgAGPAT-KO was transfected in RH-DKu80 parasites for a simple mutant DTgAGPAT and in DTgATS2 parasites to obtain a double mutant DTgATS2/DTgAGPAT. The plasmid pMORN1-CherryRFP-MORN1/SagCAT were transfected in both RH-DKu80 and DTgATS2 parasite lines.

All other transfections were performed with 50 mg of DNA and electroporation conditions were as described above. Transfected parasites were incubated at different concentration with HFF cell 48 h prior to immunofluorescence assay.

T. gondii growth assays -Plaque Assay HFF monolayers were infected with 500 parasites and allowed to develop for 10 days before staining with Crystal Violet (Sigma) and cell growth assessment by light microscopy for the presence of intact HFF. To obtain statistical assessment, each strain was grown in each condition in triplicate and the plaque area in the same square unit (n = 6) are measured. Boxplot with whiskers from minimum to maximum with median.

-Cell-based assay T. gondii growth was determined with an automatic microscope-based screening (Olympus ScanR, Japan). HFFs were seeded at a density of 10,000 cells per well into 96-well plates and were allowed to grow and equilibrate for 48 h at 37 C. Cells were then infected with 4 3 104 parasites/well. Invasion was synchronized by briefly centrifugation of plate at 250 g and placed at 37 C for 2 h. The assay was run for 30 h. Hoechst 33342 (Life technologies) stain was then loaded on live cells/parasites at 5 mg/ml for 20 min. Infected cells were fixed with PFA (3.7%) for 10 min at 37 C. A mouse anti-GRA1/Alexa488 labeling (dilution 1:500) was used to identify parasitophorous vacuoles. A total of 20 fields per well were taken using the 20X objective. Images were collected for the distinct fluorescence channels (Hoechst 33342: e.g.,(360)(361)(362)(363)(364)(365)(366)(367)(368)(369)(370). Images were then analyzed using the ScanR analysis software (Olympus, Tokyo, Japan). For Alexa488 channels images (vacuoles) an intensity algorithm module was used where a fixe threshold was defined with a minimum of 100 pixels size in order to segment the smallest vacuoles (one or two parasite). For Hoechst channel images (parasites nuclei), image process consists to apply a strong background correction and detected parasites with an edge algorithm. A minimum object size of 5 pixels and a maximum object 20 pixels larger one was chosen to discriminate each parasite. ScanR analysis module interface as in flow cytometry allow us to extract and display data as scatterplots and histograms. Using a ''gating'' procedure we were able to hierarchically filter selected data points with precise boundaries (e.g., number of vacuoles versus number of parasite/vacuoles). The proliferative index was evaluated by parasite/vacuole number ratio. To assess statistically, the samples were prepared in quadruplicate (n = 4). T. gondii egress assay WT or DTgATS2 parasites were incubated on HFF cells for approximately 26 h before aspirating medium and replacing with DMEM containing 2 mM A23187 or DMSO in quadruplicate (n = 4). Parasites were incubated for 3 min before addition of an equivalent volume of 2x fixative containing 5% Paraformaldehyde, 0.05% glutaraldehyde in PBS (final concentration 2.5% Paraformaldehyde, 0.025% glutaraldehyde). Cells were fixed for 15 min before permeabilizing with 0.025% Triton X-100 in PBS for 10 min and then Blocking overnight in blocking solution (2% FBS in PBS). Samples were then probed by immunofluorescence assay and counted manually for egress.

T. gondii Red/Green parasite invasion assay Experiment was performed as per (Katris et al., 2014). Parasites were grown for 2 days in quadruplicate (n = 4). and harvested intracellular after replacing medium with ENDO buffer (44.7 mM K2SO4, 10 mM MgSO4, 106 mM sucrose, 5 mM glucose, 20 mM Tris-H2SO4, 3.5 mg/ml BSA, pH 8.2). Cells were scraped, needle passed, filtered and centrifuged at 1800 rpm for 10 min. Cells were resuspended to a concentration of 2.5 3 107 cells ml-1 in ENDO buffer and settled for 20 min onto host cells. Once settled, medium was aspirated and replaced with Invasion buffer (DMEM, 3% FBS and 10 mM HEPES). Parasites were allowed to invade for 15 min before fixation with 2.5% Paraformaldehyde and 0.02% glutaraldehyde. Samples were then blocked in 2% FBS in PBS overnight at 4oC. Samples were probed with mouse anti-SAG1, before washing with PBS, then permeabilized with 0.25% Triton e6 Cell Reports 30, 3778-3792.e1-e9, [START_REF] Romano | The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network[END_REF] X-100 in PBS. Cells were then probed with rabbit anti-GAP45 and washed in PBS. Samples were then probed with Alexafluor antimouse 546 and anti-rabbit 488 before mounting onto slides. Cells were imaged by microscopy and invasion rate determined using ImageJ.

Plasmodium falciparum growth assays P. falciparum NF54 wild-type parasites and FabI-KO (Vaughan et al., 2009) were maintained as previously described [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]) at 2% hematocrit in RPMI-HEPES supplemented with AlbuMAX II (GIBCO). Intra-erythrocytic growth assays in standard media were performed by monitoring the replication of tightly synchronous parasites (5% sorbitol) over four asexual cycles as previously described [START_REF] Mi-Ichi | Intraerythrocytic Plasmodium falciparum utilize a broad range of serum-derived fatty acids with limited modification for their growth[END_REF][START_REF] Mitamura | Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum[END_REF]. Media was replaced daily, sub-culturing were performed every 48 h when required, and parasitemia monitored by Giemsa stained blood smears. Growth assays in lipid-depleted media were performed by synchronizing parasites, before transferring trophozoites to lipid-depleted media as previously reported (Botte ´et al., 2013;Shears et al., 2017). Briefly, lipid-rich AlbuMAX II was replaced by complementing culture media with an equivalent amount of fatty acid free bovine serum albumin (Sigma), 30 mM palmitic acid (C16:0; Sigma) and 45 mM oleic acid (C18:1; Sigma). All assays were performed in triplicates on different days.

Immunofluorescence assay and Microscopy

Parasites were infected to HFF cells grown on coverslips as previously mentioned (Amiar et al., 2016). Primary antibodies used: Mouse anti-HA antibody (Roche, 1:1000), anti CPN60 (1:1000), anti GAP45 (1:1000), rabbit anti-ACP (1:2000), rabbit anti-TOM40 (1:3000), polyclonal rabbit anti-IMC1, anti-MIC4 antibodies (1:1000), rabbit anti-Sumo21 at (1:500) and mouse anti-Sag1 (1:500), anti-LBPA (1:500) or anti-LC3 (1:500). Secondary antibodies: anti-mouse Alexa 488 or 546, anti-rabbit Alexa 546-(ThermoFisher Scientific, 1:10000). Mitotracker (1mM) was diluted in DMEM 1:5000 (100-300 nM working concentration).

For the immunofluorescence assay (IFA) parasites were grown on confluent HFF on coverslips and fixed in PBS containing 2.5% paraformaldehyde (PFA) for 15 min at room temperature (RT). Samples were permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT prior to blocking in PBS containing 3% BSA and subsequent incubation with primary antibodies then secondary antibodies diluted in the blocking solution. Labeled parasites were stained with Hoechst (1:10000, ThermoFisher Scientific) for 20 min and then washed three times in PBS before final mounting of the coverslips on a glass slide using Fluoro-Gel (Electron Microscopy Sciences). The fluorescence was visualized using fluorescence microscope (Axio Imager 2_apotome; ZEISS) with 63x objective.

Nile red staining of lipid droplets

The parasites were allowed to infect and growth in confluent monolayer HFF grown on coverslips, in the ± ATc conditions for x days and then fixed in PBS containing 2.5% paraformaldehyde (PFA) for 15 min at room temperature (RT). Samples were permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT and stained with primary rat anti-HA antibody followed by detection with secondary AlexaFluor 488-conjugated goat anti-rat antibody. Thereafter, the sample coverslips were incubated for 1 h with Nile red in 1X. Lastly, three washing steps with 1X PBS were performed before proceeding to DNA staining with Hoechst. The coverslips were mounted onto a glass slide in fluorogel vefore proceeding to imaging using fluorescence microscope (Axio Imager 2_apotome; ZEISS). For visualizing nile red stained droplets yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) was used on the axio imager. Quantification in ± ATc condition was done by counting the no. of lipid droplets per parasite.

Activity analysis in LPAAT-deficient E. coli strains Escherichia coli strain deficient in LPAAT/AGPAT activity [SM2-1 DplsC, Coli Genetic Stock Center #7587, Yale University] ( [START_REF] Coleman | Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity[END_REF] was used to confirm LPAAT activity in both TgATS2 and TgAGPAT.

Coding sequence of TgATS2 was synthesized (Genscript). TgAGPAT coding sequence was amplified by RT-PCR using primer sets 5 0 -ATGGCGTCCACGCCGCTGC-3 0 /5 0 -TTAGAGACCGTGGCCTCGGTG-3 0 and TgAGPATDN-ter1-72 coding was amplified by RT-PCR using primer sets 5 0 -CTCAACCGCCCGCCCAGGAATTA-3 0 /5 0 -TTAGAGACCGTGGCCTCGGTG-3 0 . These sequences were digested and ligated into HindIII restriction site on pQE30Xa vector (Quiagen) to generate expression vectors. Additionally, gene coding for E. coli LPAAT activity plsC, was amplified from E. coli DH5alpha genomic DNA using primer sets 5 0 -CTATATATCTTTCGTCTTAT TATTAC-3 0 / 5 0 -AACTTTTCCGGCGGCTTC-3 0 and ligated into pQE30Xa vector. Then these acyltransferase vectors and empty pQE30Xa vector as negative control were transfected to electrocompetent cells of SM2-1 DplsC deficient E.coli. pREP4 repressor vector to regulate Lac promotor activity. Transformed bacterial populations were grown at 37 C in order to promote growth of all isolates. Two independent clones of each bacterial strain that harbors each plasmid-of-interest were isolated for this study. Rescue of LPAAT activity in SM2-1DplsC mutant was measured by the ability to grow at elevated temperature, 42 C, non-permissive temperature in LB medium as previously described [START_REF] Coleman | Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity[END_REF]. Bacteria were first grown in LB media at 37 C to stationary phase, then the cultures were diluted to OD600 = 0.04 and finally inoculated with several dilutions (at 10-1 to 10-6) on LB plates and incubated for 24 h at permissive (30 C) and non-permissive (42 C) temperatures. All experiments were conducted in triplicate with both independent clones.

Transmission electron microscopy

Parasites were grown for 24 h in Labteks (Nunc, Thermofisher) before fixation in 0.1 M cacodylate buffer with 2.5% glutaraldehyde for 2 h. Samples were then kept in fixative. at 4oC until further processing. Sample were then post-fixed 1h with 1% osmium tetroxide in cacodylate buffer followed by overnight in 2% uranyl acetate in distilled water. After dehydration in graded series of acetonitrile, samples were progressively impregnated in Epon812, the wells were then filled with fresh resin and allowed to polymerize 48 h at 60 C. Ultrathin 70 nm section were obtained with a Leica UCT Ultramicrotome and collected on copper grids. Grids were post-stained with uranyl acetate and lead citrate before their observation on a Jeol1200EXII Transmission Electron Microscope. All chemicals were from Electron Micrsoscopy Sciences.

Lipidomic analysis by GCMS extraction from T. gondii tachyzoites

Lipid extraction and analysis of tachyzoites was performed as previously described (Ramakrishnan et al., 2012;Amiar et al., 2016;Dubois et al., 2018). Freshly egressed tachyzoites (1 3 10^8 cell equivalents) grown in standard culture (n = 4) or in starvation culture (n = 3), were metabolically quenched by rapid chilling of the cell suspension in a dry ice/ethanol bath and lipids were extracted in chloroform/methanol/water (2:1:0.8, v/v/v containing 25 nmol tridecanoic acid C13:0 as extraction internal standard) for total lipid analysis.

-For lipid quantification Total lipid extraction was performed as described previously (Amiar et al., 2016). Parasites were prepared as described above except for the addition of 0.1 M HCl to promote PA and LPA extraction. Pooled organic phase was subjected to biphasic separation by adding 0.1 M HCl. In both protocols, the organic phase was dried with speed vaccum and dissolved in 1-butanol.

-Total lipids analysis An aliquot of the lipid extract was dried in vacuum concentrator with 1 nmol pentadecanoic acid C15:0 as internal standard. Then the dried lipid was dissolved in the chloroform/methanol, (2:1, v/v) and derivatised with MethPrep II (Alltech). The resulting fatty acid methyl esters was analyzed by GC-MS as described previously (Amiar et al., 2016). Fatty acid methyl esters were identified by their mass spectrum and retention time compared to authentic standards. Lipid data was analyzed using Agilentâ Masshunter software.

-Lipid quantification Total lipid fraction was separated by 2D-HPTLC (Merck) with 5 mg PA(C17:0/C17:0) and 5 mg LPA(C17:0) (Avanti Polar lipids) using chloroform/methanol/28% NH4OH, 60:35:8 (v/v) as the 1st dimension solvent system and chloroform/acetone/methanol/acetic acid/water, 50:20:10:13:5 (v/v/v/v/v) as the 2nd dimension solvent system (Amiar et al., 2016). For DAG analysis, total lipid fraction was separated by 1D-HPTLC using hexane/diethlether/formic acid, 80:20:2 (v/v/v) as solvent system. The spot on the HPTLC corresponding to each lipid was scrapped off and lipids were directly derivatised with 0.5 M methanoic HCl in the presence of 1 nmol pentadecanoic acid (C15:0) as internal standard. The resulting fatty acid methyl esters were extracted with hexane and analyzed by GC-MS (Amiar et al., 2016). Resulted FAME and cholesterol-TMS was analyzed by GC-MS (5977A-7890B, Agilent). FAME was then quantified using Mass Hunter Quantification software (Agilent). All statistical analyses were conducted using GraphPad Prism software. P values of % 0.05 from statistical analyses (Ttests) were considered statistically significant.

Stable isotope labeling of T. gondii Stable isotope labeling using U-13 C-glucose (Cambridge Isotope Laboratories, USA), lipid extraction, and GC-MS analysis was performed as previously described in Ramakrishnan et al. (2012) and Amiar et al. (2016). Freshly infected HFF were incubated in glucose-free medium supplemented with 8 mM U-13 C-glucose. For FBS starvation study, 5% FBS was add to U-13 C-glucose medium in standard culture conditions and 1% FBS was add to U-13 C-glucose medium in starvation culture condition. Parasites were harvested 72 h post-infection and metabolites extracted as above.

Phospholipid import assay

Freshly lysed cultures of WT or DTgATS2 parasites (n = 3) were harvested, filtered and resuspended in DMEM to a concentration of approximately 2 3 108 cells ml-1. Cells were then mixed with a 2x solution containing 10 mg ml-1 NBD-PA or NBD-PC (5 mg/mL final) and incubated at 37oC. Parasites were then spun down, resuspended in PBS. PFA was then added to a final concentration of 2.5%, and cells were fixed for 15 min before being spun down again and resuspended in 1xPBS. Parasites were smeared onto polyethyleneimine coated coverslips, and then probed with anti-SAG1 primary (1:1000) and anti-mouse Alexa 546 secondary antibodies (1:10000) by immunofluorescence microscopy, stained with DAPI (1:10000) and mounted onto slides. Samples were imaged by microscopy. SAG1 labeling was used to identify parasites using ImageJ and then estimate the amount of NBD-lipid uptaken by the parasites.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses for all experiments were performed with Prism software v7 (GraphPad). In experiments comparing only two groups, t test with Holm-Sidak correction were used to compare the experimental group with the control group. For other experiments including 3 groups, non-parametric ANOVA tests (Sidak correction for multiple tests) were used. Individual p values are indicated in each figure. Each experiment was done in n = 3 otherwise mentioned in material metthods. For lipidomic analysis, e8 Cell Reports 30, 3778-3792.e1-e9, [START_REF] Romano | The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network[END_REF] Introduction:

Apicomplexa are unicellular eukaryotes, which most organisms are obligate intracellular parasites. The Apicomplexa phylum comprises important human pathogens such as Toxoplasma gondii causing toxoplasmosis and Plasmodium spp., the causative agent of malaria. These pathogens represent a global human and social threat against which there is no efficient vaccine and which are becoming increasingly resistant to all marketed drugs, especially in the case of Plasmodium falciparum, the major agent of lethal human malaria (1).

There is a pressing need for the identification of new drug targets and for the development of novel inhibitors.

Understanding the complex metabolic pathways by which these parasites can obtain the nutrients essential for their survival is an important avenue for drug development.

Specifically, lipid synthesis is a pivotal and essential pathway for the parasite during its intracellular development for membrane biogenesis, proper lipid homeostasis and lipid signalling. Due to its complex and unique evolution, the lipid synthesis pathway is highly compartmentalized and forms a puzzle pathway with enzymes of different origins. Indeed, most Apicomplexa (to the exception of Cryptosporidium) harbour a relict non-photosynthetic plastid named the apicoplast (Apicomplexa plastid) (2,3), which has been acquired by the secondary endosymbiosis of a red algal ancestor (4). Similarly as in plant and algal plastids, the apicoplast contains a prokaryotic type II fatty acid synthesis pathway, or FASII pathway, that is essential in both T. gondii and P. falciparum (5)(6)(7)(8)(9)(10). The apicoplast also has the capacity to use FASII fatty acids and form lysophosphatidic acid (LPA) and phosphatidic acid (PA), which are crucial precursors used for the bulk membrane biogenesis and for the survival of the parasite (8,(10)(11)(12)(13)(14)(15)(16). Apicoplast-generated fatty acids and LPA are then exported towards the endoplasmic reticulum where they can be elongated and desaturated by elongases and dehydratase to expand the FA range used to maintain the parasite lipid homeostasis in each of its intracellular compartment (7,15,17).

In addition to these de novo lipid synthesis pathways, parasites are also capable of scavenging lipids and other resources from the host and the external environment. The parasite lipid synthesis, composition and homeostasis depend on a fine-tuning between the de novo synthetic pathways, the scavenged lipid moieties and the trafficking of these lipids.

Furthermore, parasites can sense the availability of lipids and other nutrients from the environment to modulate the balance between de novo and scavenging metabolic pathways and thus maintain membrane biogenesis, proper growth, division, and thus survival and pathogenesis (18)(19)(20).

Acetyl co-enzyme A (acetyl-CoA) is a crucial metabolite in the central carbon metabolism of Apicomplexan parasites, including lipid synthesis, and the mitochondrial TCA cycle (7,(21)(22)(23)(24)(25). Indeed, the main precursor of the apicoplast FASII is acetyl-CoA, which is generated via the apicoplast pyruvate dehydrogenase (PDH) (6). The apicoplast PDH is fuelled via the import of phosphoenolpyruvate, which is generated via the cytosolic glycolysis pathway and then transported via the apicoplast phosphate transporter (APT, also named the apicoplast triose phosphate transporter in P. falciparum (7,23), in a similar manner as in plant plastids (26)(27)(28)(29). The ER fatty acid elongation pathway also requires acetyl-CoA as a carbon source to elongate the apicoplast generated fatty acids. Unlike plants and most eukaryotes, Apicomplexan mitochondria lack a canonical PDH to synthesise Acetyl-CoA, essential for the TCA cycle (21). Instead, the parasite uses a Branched-chain Keto acid dehydrogenase (BCKDH) that possesses a dual function to also make acetyl-CoA for the TCA cycle (21,30). In T. gondii, acetyl-CoA can also be made via a cytosolic ATPcitrate lyase (TgACL) using a by-product of the TCA cycle, oxaloacetate, unlike P. falciparum that seems to lack a homolog of TgACL. However, both T. gondii and P. falciparum possess a single acetyl-CoA synthetase (ACS) that can use acetate imported from the host and/or the external environment to for acetyl-CoA by active transfer of acetate to the Co-enzyme A (CoA) (22). Recent analysis of the T. gondii ACS and ACL showed that each enzyme is dispensable alone but that the dual knockout parasite strain for TgACS and TgACL is not viable (30). Taken together these data point at the importance of acetyl-CoA and its synthesis in the metabolism of these parasites. To date, no metabolomic analysis has been performed to unravel the metabolic role of TgACL and especially TgACS, which is the sole enzyme capable of using a scavenged substrate, i.e. acetate, for acetyl-CoA synthesis.

Here we generated an inducible knock-down of the T. gondii ACS, TgACS-iKO. We conducted state of the art stable isotope labelling, using 13 C-Uglucose and 13 C-U-acetate, combined to mass spectrometry-based lipidomic analysis of the TgACS-iKO to determine its putative role in fatty acid synthesis and fatty acid elongation, respectively. We report that the enzyme contributes to the parasite lipid synthesis pathways, specifically for the elongation of fatty acid by the ER elongases whereas it does not participate in the de novo synthesis pathways for fatty acid synthesis. This is the first report that TgACS has a role beyond being a source of acetyl-CoA for protein acylation and histone acylation and shows the versatility of the parasite to confront its metabolic demand and nutrient availabilities.

Results

TgACS is a cytosolic protein that is not essential during tachyzoite stage.

In order to identify the role of acetyl-CoA synthetase (ACS), we first searched for a candidate ACS in T. gondii genome using the ToxoDB website (http://toxodb.org/toxo/). We found a predicted gene annotated as ACS (accession number: TGGT1-266640) based on sequence homology with other characterised ACSs (31)(32)(33). The candidate protein was bearing typical domains for ACS such as AMP binding (PF13193) and CoA binding (TIGR02188). Comparison of TgACS protein sequence against the one of Salmonella enterica, SeACS, which protein was crystallized (34), showed a high level of conservation between the two proteins. Indeed, alignment showed a 52% identity over 91% of the protein covered when comparing SeACS to TgACS. Most residues involved in CoA binding, (i.e. F222, A223, G224, R250, K253, I255, A417, S585, G586, H587, R588, K642, R646) and AMP binding (i.e. I370, V446, T472, Y473, W474, 8475, T476, E477, I574) (Fig1. A) are identical or highly conserved as well. Furthermore, we searched for homologs of ACS in discernable alteration in plaque size so as ATc treatment of the parental lines produced no detectable growth retardation (Fig. 3CD). This suggested that TgACS is not essential in accordance with a recent genome wide analysis of essential genes in Toxoplasma by CRISPR-Cas9 knockout, which pointed at the likely non-essential role of TgACS (38).

TgACS disruption slightly alters the total fatty acid content of the parasite.

Since the ACS gene encodes for an enzyme to produce acetyl-CoA, a major precursor for fatty acid synthesis and fatty acid elongation, it is possible that loss of TgACS may alter the lipid profile of the parasite. We first qualified and quantified the fatty acid content in the TgACS-HA-iKO parasite in the absence and presence of ATc for 4 days. Fatty acid moieties of glycerolipids were derivatised to fatty acid methyl ester (FAME), then FAMEs were quantified by gas chromatography-mass spectrometry (GC-MS, 5977A-7890B, Agilent Technology). There was no major difference in total fatty acid content between the reference and induced TgACS knockout strains (Fig. 4A). However, slight differences in the fatty acid composition could be detected (Fig. 4BC). Although TgACS can generate acetyl-CoA that is hypothesized to be used for the elongation of fatty acids, there was slight yet non-significant decrease in the longer chain fatty acids amount known to be produced via the elongation pathway (7) such as C20:1, C22:1, C24:1. Interestingly, however, in the ATc treated parasite, the apicoplast FASII-generated C12:0 and C14:0 were decreased significantly, while C22:6, a fatty acid source believed to be scavenged from the host, was significantly increased.

TgACS disruption does not impair apicoplast fatty acid synthesis.

To determine the potential role of TgACS for fatty acid synthesis, including de novo fatty acid synthesis in the apicoplast and FA elongation in the ER, we performed metabolic labelling on ATc-treated and untreated TgACS-iKO-HA parasites with stable isotope precursors, i.e. 13 C-universally labelled carbon substrate, U-13 C-glucose or U-13 C-acetate, respectively. These substrates can be incorporated to the parasites and are used for the fatty acid synthesis in different pathways (7,15,39). Incorporation of 13 C-glucose to fatty acids determines the de novo synthesized fatty acids via FASII in apicoplast (7,15,39), whereas the incorporation of 13 C-acetate to fatty acids determines the elongation of fatty acids in cytosol (7,17). The resulting labelled fatty acids can be distinguished by the shift of mass by GC-MS. The degree of the incorporation of 13 C into fatty acids (%carbon incorporation) is determined by the mass isotopomer distribution (MID) of each FAMEs. These together can delineate the exact effect on lipid metabolism of TgACS knockdown. MID can be obtained by guest, on April 22, 2018 This slight reduction of relative abundance for the M+14 (and M+12) could be a sign of slight reduction of the FASII activity.

TgACS disruption significantly alters endoplasmic reticulum fatty acid elongation pathway

To investigate if acetyl-CoA generated by TgACS is involved in the elongation of fatty acid, we first labelled parasites with U-13 C-acetate continuously for 4 days with or without ATc (Fig. 6). In this condition, there was incorporation of 13 C label to all fatty acids analysed in both with or without ATc. Interestingly, the 13 C incorporation from acetate to shorter fatty acids, C14:0, C16:0 in the TgACAS-HA-iKO with ATc was significantly higher than that without ATc (Fig. 6A). In the meantime 13 C incorporation to longer fatty acids known to be products of the elongation pathway, especially C18:1, C20:0 and C20:1, was significantly reduced (Fig. 6A). MID analysis of 13 C integration into C14:0 and C16:0 showed that the measured labelling increase displayed an unusual pattern of M+1 incoporation from M+2 to M+8 (Fig. 6BC). However, there was no sign of two by two increase of the mass, which was seen in the U- 13 C-glucose as signature of apicoplast FASII suggesting acetate was not directly incorporated into the apicoplast as a substrate for FASII but rather that acetate was catabolised to a single carbon molecule and then metabolised to be used in FASII pathway. MID analysis of C20:0 did not show clear increase of M+2, M+4, a usual signature for the elongation pathway (Fig. 6D). Although 13 C incorporation from acetate was altered, there was no total abrogation of such incorporation. Taken together, this also suggest 4 days of incubation with U-13 C-acetate with or without ATc was likely too long and may not be the best suited approach to determine effect of the loss of TgACS.

We thus treated parasites with ATc for two days prior to addition of U-13 C-acetate and grown for a further two-days (pre-treatment 2 days) to suppress most of TgACS presence in the parasite. In this condition, 13 C integration from 13 C-acetate was almost abrogated in most long fatty acid chains C18:0, C20:0, C22:0 and C24:0 generated via the elongation pathway (Fig. 6E). Here, no incorporation of 13 C to C14:0, C16:0 in both strains was observed (Fig. 6FG) suggesting that what observed in Fig. 6BC was probably due to the catabolism of acetate due to long incubation in the course of TgACS deactivation. The MID analysis of C20:0, showed a clear increase of M+2, and M+4, in the absence of ATc (i.e. wild type condition), which is the signature of elongation of the FASII products C18:0 and C16:0 (15). However, in the presence of ATc, i.e. lack of TgACS, labelled C20:0 did not contain any increase of M+2 and M+4 (Fig. 6H). This clearly suggests that the loss of TgACS could not provide the substrate, acetyl-CoA for the elongation of fatty acids. Similar results were measured for the other altered FA chains, suggesting that C18:0 is elongated from C16:0, C22:0 from C18:0 and C16:0, C20:1 predominantly from C18:1mainly and slightly from C16:1/0 and C24:0, C16:0 and C14:0 (Fig. S2). Collectively these metabolic results indicate that the loss of TgACS leads to a reduction in the elongation of FA by the lack of acetyl-CoA it produces for the pathway.

Discussion:

In this study we determined the role of the sole enzyme capable of generating acetyl-CoA from a scavenged substrate (i.e. acetate) for lipid synthesis in T. gondii. Indeed, acetyl-CoA is a central metabolite that is involved in the TCA cycle, fatty acid synthesis, fatty acid elongation and post-translational protein acylation, including histone acylation for genetic regulation. The apicoplast FASII pathway generates its own pool of acetyl-CoA via the apicoplast PDH and the mitochondrial BCKDH generates the acetyl-CoA required to initiate the TCA cycle (30). The source and role of acetyl-CoA for cytosolic fatty acid synthesis via the FASI pathway and the endoplasmic reticulum elongation pathway remain to be fully understood. Previous studies showed that T. gondii possesses two enzymes capable of generating acetyl-CoA: the acetyl-CoA synthetase (TgACS) and the ATP-citrate lyase (ACL) (30). Where TgACS is theoretically capable of using acetate and binds it to CoA, TgACL uses existing citrate from the TCA to generate acetyl-CoA. Interestingly the localization of TgACS shows a uniform cytosolic and nuclear localization, as previously described (30,37).

The nuclear localization supports the potential role of TgACS for providing acetyl-CoA in the nucleus for histone acetylation. Further analysis to determine its putative role in histone modification and chromatin remodelling by determining T. gondii acetylome on the TgACS-iKO mutant would be important to conduct. Here, we disrupted TgACS and were able to determine its role in generating acetyl-Coa for fatty acid biosynthesis.

The loss of TgACS disrupts the typical mechanism of FA elongation, which relies on three elongases together essential for parasite survival (7). Here our results clearly demonstrate that the acetyl-CoA generated by TgACS is used by the elongases to contribute to the production of many elongated FA initially produced by the apicoplast FASII (C18:0, C18:1, C20:0, C20:1, C22:0, C24:0) (Fig. 7, 15). The lack of major changes in the FA composition and the non-lethal phenotype of TgACS-iKO mutant clearly support the presence of another source of acetyl-CoA for the essential FA elongation pathway. This is likely a redundant role of TgACL as previously shown when both TgACS and TgACL were knocked out, which eventually killed the parasite. (30)This hypothesis is also strengthened by the increase of ACL abundance when TgACS is disrupted (30). Interestingly, P. falciparum seems to lack a homolog to the citrate lyase and possesses the sole ACS to generate acetyl-CoA from acetate (22), which seems essential for the parasite (40). Further analyses are a prerequisite to understand the differences between T. gondii and P. falciparum lipid synthesis and use of acetyl-CoA.

Genetic ablation of TgACS does not kill the parasite; however, metabolic plasticity enables continued parasite survival via providing alternate substrate options maintaining elongation at a suitable level as measured by the absence of major changes in the total FA composition. As an evidence of such compensation, upon the loss of TgACS, C12:0 and C14:0, major products of FASII (15), decrease, whilst C22:6, a FA chain believed to be scavenged (7,41), increases. When we incubated parasites with 13 C-U-acetate for 4 days, the incorporation of labelling was observed in both TgACS-iKO with or without ATc although 13 C-U-acetate was not supposed to be used as substrate for the de novo fatty acid synthesis, i.e. incorporation to short chain fatty acids, C14:0 fatty acids. In addition, the labelling in C14:0 was more abundant upon the loss of TgACS suggesting that parasite enhanced some metabolic pathway and used acetate for de novo fatty acid synthesis upon the loss of TgACS.

First of all, it should be noted that both TgACS-iKO with or without ATc had incorporation of 13 C-U-acetate to C14:0 only up to M+7 and to C16:0 only up to M+8 in each masses. This means only one carbon of acetyl-CoA was labelled with 13 C, therefore parasites have only half of number of fatty acid chain is labelled in continuous manner without two by two steps. This was only observed when parasites were incubated for four days with 13 C-U-acetate with or without ATc but not in two days even in the -ATc condition. This suggests that acetate was not directly used for the TCA cycle similarly shown in Plasmodium, that 13 C labeled acetate is rapidly incorporated to acetyl-CoA and then to acetylated alanine and acetylated glutamate but not to keto-glutarate, TCA intermediate (22). It is possible that acetate was recycled from those acetylated amino acid to be metabolized to pyruvate probably via TCA cycle to serve a precursor for the de novo fatty acid synthesis. Another potential explanation for these is the un-used acetate was catabolised to give single carbon molecule, bicarbonate and CO 2 to be used in another metabolic pathway. Finally, T. gondii is able to generate propionyl-CoA from the degradation of leucine, isoleucine, and valine (42). This propionyl-much influence over the parasite FA composition but (d) that it provides acetyl-CoA for the elongation of FA, and actively participates to this pathway, essential for parasite survival (7).

These data show for the first time that one of the enzymes capable of the synthesis of Acetyl-CoA is also involved in the parasite lipid synthesis and the plasticity of T. gondii to compensate for the loss of proteins participating in these crucial pathways for membrane biogenesis.

Materials and Methods

Sequence analysis and structure generation

TgACS gene (TGGT1-266640) was identified by using toxoDB (http://toxodb.org/toxo/) and alignment with Salmonella enterica, was performed on Multalin (http://multalin.toulouse.inra.fr/multalin/multalin.html (49). The 3D structure was determined via protein threading against the previously crystallized S. enterica ACS, (34) using SwissProt bioinformatics (https://swissmodel.expasy.org/).

T. gondii strains and cultures

T. gondii tachyzoites (RH-∆Ku80 TATi (36) and TgACS-iKO-HA) were maintained in human foreskin fibroblasts (HFF) using Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 1% foetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and 25 µg/mL gentamicin (Gibco) at 37°C and 5% CO 2 .

Construct design

TgACS (TGGT1-266640) gene sequences was obtained from ToxoDB. The TgACS open reading frame was PCR amplified using primers 5'-GAAGATCT ATGGAGAAAGATAGGAACACTATGGAGGG and 5'-. TGGCCTAGGAGCTTTCGCA-AGAGAGCCCC, TgACS un-translated flanking regions were generated using the following primers, 5' UTR 5'-GGAATTCCATATGTACTTTCCACATACGTCTGCTTGTGC and 5'-GGAATTCCATATGGGTGTTCCTGGTTCTGAAATGTTGC and the 3'UTR with 5'-AT-ACCCGGGACGATTTATACACATGGTTAGACCAGGC and 5'-ATAAGAATGCG-GCCGCACGTCCTTCATTAGCCATCTGTTGC. PCR was performed using PrimeSTAR Max DNA Polymerase (TaKaRa, Japan) denaturing at 98ºC for 10 s and annealing at 60ºC for 10 s and extension at 72ºC (1 min/kbp). These PCR products were then inserted into the vector pDt7s4H (Fig. 1B) [START_REF] Agrawal | Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins[END_REF][START_REF] Agrawal | Rice octadecanoid pathway[END_REF]). The resulting construct was transfected into T. gondii RH Ku80_TATi strain parasites as described (15). Transfected parasites were then selected on

Antibodies and immunofluorescence assays

Primary anti-CPN60 (rabbit) antibodies were used at a dilution of 1/6000, anti-HA at 1/1000 (Mouse, Invivogen). Secondary AlexaFluor 488-and 546-conjugated anti-mouse and anti-rabbit antibodies (Life Technologies) were used at 1/10000. Parasites were grown on confluent HFF on coverslips and fixed in PBS containing 4% paraformaldehyde for 30 min, at room temperature (RT). Samples were permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT before blocking in PBS containing 3% BSA and incubation with primary antibodies then secondary antibodies diluted in the blocking solution. Labelled parasites were stained with Hoechst (1/10000, Life technologies) for 20 min and then washed three times in PBS then H 2 O. Coverslips were mounted onto slides prior to observation using an epifluorescent microscope (Zeiss, Germany).

Western blotting

Protein expression was analysed by western-blot on freshly egressed parasites. Equal amount (50 µg) of protein were boiled in SDS-PAGE buffer separated on a 4-12% gradient SDS-polyacrylamide (Life Technologies) and transferred to PVDF membrane (Millipore) using the XCellII Blot Module (Invitrogen). The membrane was blocked with skim milk and then probed with monoclonal mouse anti-HA antibodies (InvivoGen) at 1:2000 and mouse anti-Gra1 antibodies at 1:3000. Secondary Goat anti-mouse HRP conjugated antibodies (Thermo Scientific) were used at 1:20000. Signal was detected after membrane staining with Luminata Crescendo Western HRP detection kit (Millipore).

Phenotypic analysis

Plaque assays were performed with 500 parasites infected to HFF confluent monolayers in culture flasks (25 cm 2 ). TgACS-HA-iKO parasites were grown in the presence or absence of ATc 0.5 µg/mL for 10 days. Then cells were fixed with ethanol and followed by staining with Crystal Violet (Sigma).

Lipid extraction from T. gondii tachyzoites and gas-chromatography mass spectrometry analysis

Intracellular tachyzoites (1 × 10 8 cell equivalents per replicate) were harvested after metabolic quenching in dry ice-ethanol (100%) to rapidly stop the metabolism as previously described (7,[START_REF] Macrae | Mitochondrial Metabolism of Glucose and Glutamine Is Required for Intracellular Growth of Toxoplasma gondii[END_REF]. Then total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v containing 25 nmol tridecanoic acid (C13:0) as internal standard for extraction) for 1 h at 4 o C, with periodic sonication. Then polar and apolar metabolites were separated by phase partitioning. For lipid analysis, the organic phase was dried under N 2 gas and dissolved in chloroform/methanol, 2:1 (v/v). Then the lipid was mixed with 1 nmol pentadecanoic acid (C15:0) as internal standard and derivatised using MethPrep II (Alltech). The resulting fatty acid methyl esters were analysed by GC-MS as previously described (7,[START_REF] Macrae | Mitochondrial Metabolism of Glucose and Glutamine Is Required for Intracellular Growth of Toxoplasma gondii[END_REF]. All fatty acid methyl esters were identified by comparison of retention time and mass spectra from GC-MS with authentic chemical standards. Then fatty acid methyl esters were normalized to cell number and extraction efficiency and quantified. The experiments were repeated as indicated using each independent mutant line as a biological replicate.

Stable isotope metabolic labelling of T. gondii fatty acids.

Stable isotope metabolic labelling experiment using U-13 C-glucose or U-13 C-acetate (Cambridge Isotope Laboratories, USA) followed by lipid extraction and GC-MS analysis was performed as previously described (7,15,[START_REF] Macrae | Mitochondrial Metabolism of Glucose and Glutamine Is Required for Intracellular Growth of Toxoplasma gondii[END_REF]. Parasites were infected to confluent HFF and incubated in the presence or absence of ATc (2 µM, Sigma-Aldrich). For the change of 13 C labelling in the course of TgACS-HA suppression by ATc, glucose-free medium supplemented with a U-13 C-glucose or U- 13 C-acetate at a final concentration of 8 mM was used concomitant with the inoculation of parasites, and parasites were harvested after four days. For the change of 13 C labelling after the loss of TgACS protein by ATc, parasites were grown for 72 h in the normal media. Then medium was replaced with glucose-free medium supplemented with 13 C-U-carbon source (concentration as above) while presence or absence of ATc is maintained. 24 h the incubation with 13 C source medium, parasites were harvested for the lipid analysis as above. Other supplements (glutamine, sodium bicarbonate, and foetal bovine serum) were added according to normal culture conditions, minimal concentration of

Introduction

Microtubules (MTs) are hollow cylindrical polymers composed of α-β tubulin heterodimers. These highly dynamic assemblies organize the cytoplasm during interphase and form the mitotic spindle to segregate condensed chromosomes during mitosis. Microtubule organization shows a remarkable diversity in eukaryotes, with striking differences in clades deriving from photosynthetic ancestors. Animal microtubules are anchored on a structured microtubule-organizing center such as the centrosome, or in many differentiated animal cells they are arranged in non-centrosomal arrays that are non-radial [1]. In contrast, in vascular plant cells that lack a structurally defined microtubule-organizing center, interphase MTs are always organized into linear bundles that assume different configurations depending on the cell type [2,3]. In Apicomplexa single-celled eukaryotes, deriving from photosynthetic ancestors, although now lacking photosynthesis [4], such as Toxoplasma gondii, microtubule organization varies during the parasite life cycle. At the tachyzoite replicative stage, a corset of 22 evenly spaced sub-pellicular microtubules, anchored to the apical polar ring, critically directs the polarized and elongated shape of the zoite. In addition, this parasite builds an unusual microtubule-containing structure at the apical tip, which is named conoid [5]. In Plasmodium falciparum, a longitudinally oriented array of two-three sub-pellicular microtubules contributes to the shape and integrity of the parasite [6].

While α and β-tubulin are highly conserved proteins, the effects of microtubule-binding drugs vary in organisms belonging to distinct evolutionary groups. For example, plant tubulin and Apicomplexan tubulins have a much lower affinity for colchicine than animal tubulin [7]. In contrast, small synthetic molecules such as dinitroanilines (oryzalin, ethafluralin or trifluralin) bind specifically plant and Apicomplexa tubulins but not vertebrate or fungi ones [8][9][10][11]. Due to their selectivity towards plant tubulin, dinitroanilines have been used as herbicides for more than 40 years [7] and represent promising leads for the design of antiparasite drug candidates in particular in the case of P. falciparum and T. gondii [9,12].

Computational methods have provided evidences that the dinitroaniline binding site of T. gondii α-tubulin is located beneath the H1-S2 loop [13]. Such a location predicts the disruption of protofilament interactions in the microtubule lattice upon dinitroaniline binding.

Besides dinitroanilines and their derivatives, no chemical entities that selectively target tubulin of plants and parasites have yet been described. This is not the case for mammalian tubulin, which is the target of numerous diverse chemical compounds [14][15][16]. Therefore, to identify new chemical scaffolds that could be used as template for novel anti-parasitic drugs or herbicide, we have designed an integrated multi-step strategy. First, a differential in silico screen of small molecules from chemical libraries, docking to the α-tubulin dinitroaniline-binding site, was performed to select compounds that bind selectively to plant/parasite tubulins. The selected compounds were then screened on plant cells using a miniaturized assay. The compounds active on the plant cell MT cytoskeleton were further tested on plantlets viability and counter screened for their effect on the human cell cytoskeleton. A few residual molecules, active on the plant cell cytoskeleton and plantlets, but showing no detectable effect on human cells, were finally tested for their effect on in vitro tubulin assembly of plant versus mammalian tubulin. The combination of these approaches picked out three active molecules that are selectively active on plant tubulin. Remarkably, two of them are structurally different from dinitroanilines, and therefore represent novel scaffolds that serve as leads for the design of new generation herbicides. Additionally, we checked whether any of the retained candidates affect T. gondii and P. falciparum growth and survival within their relevant human host cells. One of these compounds showed a low but selective toxicity on the proliferative stages of T. gondii and P. falciparum, as compared to human cells, highlighting the usefulness of such a multidisciplinary approach to discover new classes of herbicidal molecules with potential anti-Plasmodium and anti-Toxoplasma properties.

Results

2.1. Determination of 3D Discriminating Conformations of P. falciparum α-Tubulin for In Silico Screening α-Tubulin is a highly conserved protein (Figure S1). While tubulin structures have been obtained in multiple organisms and are available in the PDB database, the resolution level was not sufficient to be directly used as templates for the present differential in silico docking experiments. To perform a virtual screening on a domain conserved only in the photosynthetic lineage, in broad sense, we first selected a representative tubulin structural model in an Apicomplexa, well known to be non-photosynthetic today but deriving initially from a photosynthetic ancestor [17,18]. The sequence of P. falciparum α-tubulin (Uniprot accession: CAA34101) was thus used, focusing on regions conserved in plants. P. falciparum α-tubulin structure was determined by homology modeling [19] using bovine (AAX09051) and porcine (P02550) α-tubulin crystal structures as templates (Figure S2,step1). In the predicted structure, the H1-S2 loop (residues 35-60) locked the oryzalin-binding site, preventing molecules from penetrating inside. An early version of the conformational sampling tool S4MPLE [20] specifically operating on the torsional degrees of freedom only [21] was used to explore alternative putative poses of that loop. Main chains and side chains of the loop aminoacids, as well as side chains of residues putatively in contact with loop residues were declared mobile, while freezing the rest of the protein to its initial geometry. In order to sample a protein loop anchored to a rigid protein core at both ends, S4MPLE (Sampler for Multiple Protein-Ligand Entities, an algorithm designed for the conformational sampling of small molecules and in-silico docking experiments) needs an input of a user-chosen identifier of an existing main chain bond (here, the N-C α of the loop-central aminoacid, i.e., between residues 28 and 47), which will be formally considered as "broken". This allows free movement of the formally disjoined loop moieties in S4MPLE, while accounting for the complete molecular Hamiltonian (based, in that version of S4MPLE, on the CVFF force field [22]), i.e., including the concerned bond stretching and associated valence angle bending terms. This "trick" ensures a full sampling of possible loop geometries, while selecting only those that are properly closing the artificial "gap" and providing consistent geometries for the covalent elements. Since all other bond length and valence angle values were not subjected to changes (and remained set to their input values), the chirality of the C α involved in the formally broken bond was implicitly conserved. Several independent simulations of the system were run, using a genetic algorithm-based sampling strategy, for 1,000 generations each, until it was observed that, for 10 successive simulations, no absolutely lower energy value could be attained. We selected 100 conformers among the more stable ones according to a criterion of diversity, measured mainly on torsional axes (Figure S2, step 2). As shown in Figure 1, these conformers (Figure 1B, lower panel) present a well-formed "dockable" cleft as compared to the initial homology-modeled geometry (Figure 1B, upper panel). To select the more likely conformations, we then docked on these 100 conformers a reference set of 37 molecules comprising 10 dinitroaniline derivatives known to target the dinitroaniline-binding site [13] and 27 compounds never reported for binding tubulin, to our knowledge. By ranking the To select the more likely conformations, we then docked on these 100 conformers a reference set of 37 molecules comprising 10 dinitroaniline derivatives known to target the dinitroaniline-binding site [13] and 27 compounds never reported for binding tubulin, to our knowledge. By ranking the conformers according to their ability to discriminate between the active/non active molecules we could select the five more likely conformations of the H1-S2 loop (Figure S2, step 3).

These five conformations as well as the mammalian and the original Plasmodium tubulin conformations were the final targets for the in silico differential screening.

In Silico Selection of Compounds that Bind to the Dinitroaniline Site of α-Tubulin from the Photosynthetic Lineage

Using the five most promising conformations, a virtual library of more than 300,000 chemical compounds was screened for its ability to dock into the identified cleft. We first analyzed the FlexX scores in the whole docked database to evaluate the possibility to rank conformations based on these scores. This analysis showed that the active compounds previously reported in the literature were close to the average value in the score distribution and not among the best scores. The use of FlexX scores was thus considered as not discriminant enough to select molecules. Post-processing of docking outputs through analyses of the three dimensional protein-ligand binding interactions has been reported to be a powerful alternative strategy [23][24][25]. A thorough analysis of the interaction data obtained for known active compounds on the 100 conformers described above revealed that numerous residues involved in the interactions were located at the pocket entrance, whereas the residues located in the depth of the pocket were more rarely involved. Among them, three residues (Ser6, Ile235 and Leu167, Figure S1) were involved in interactions for every conformation (Figure 1B). We decided to use them as a criterion for selection.

Based on the protein molecule interactions, 3,023 molecules that, in one or more of the five "open" protein conformations, had one or more docking poses in interaction with Ser6, Ile235 or Leu167 were selected. We checked that, among these compounds, none had been selected for their effect on mammalian cell microtubules, in previous screening campaigns [15,16]. This was never the case, therefore validating the proposed specificity of the binding pocket for the photosynthetic lineage. Among these 3,023 compounds, 82 molecules were readily available in the laboratory and therefore picked for further in vitro analysis.

Selection of Compounds Active only on Plant Cell Microtubules

The selected compounds were tested for their effect on tobacco BY-2 cell microtubules using immunofluorescence. Image-based screening is frequently performed on adherent mammalian cells [16,26] but has never yet been achieved on plant cells. We thus developed an optimized immunostaining procedure in 96-well plates, as described in the Material and Methods section and captured images using an automated fluorescent microscope.

The effect of a two-hour incubation with each of the 82 molecules (50 µM) was compared to those of dinitroaniline (25 µM) that depolymerizes plant MTs, and colchicine (2 µM) that has low affinity for the plant tubulin. As expected, MT depolymerization was observed upon dinitroaniline treatment whereas colchicine did not affect the plant-cell microtubule network (Figure 2). Eleven compounds, out of the 82 compounds tested, were found to have an effect on cortical MTs (Figure 2). This indicates that these compounds could be able to cross the BY-2 cell membrane to impact cellular microtubules. Their effect on mammalian cell microtubules was then tested by immunofluorescence using HeLa cells. When applied at concentrations ranging from 3 to 50 µM for 2 h at 37 • C none of the compound, except compound CM872, induced detectable effects on microtubules. Compound CM872 showed a toxic effect that was detected at the higher concentration tested only, i.e., 50 µM (Figure 3). Overall, 10 compounds were found to be active only on BY-2 cell MTs with no detectable effect on HeLa cell MTs.

In order to know if these compounds could penetrate into a whole plant organism and be active, they were finally tested for their effect on Arabidopsis thaliana growth. As shown in , three compounds, i.e., compounds CM571, CM094, and CM852 inhibited plant growth. In addition, severe symptoms of chlorosis could be detected for compounds CM571 and CM852, indicating that they circulated in plant vascular tissues and induced a systemic response (Figure S3). To select the more likely conformations, we then docked on these 100 conformers a reference set of 37 molecules comprising 10 dinitroaniline derivatives known to target the dinitroaniline-binding site [13] and 27 compounds never reported for binding tubulin, to our knowledge. By ranking the

Selected Compounds Target Plant but not Mammalian Tubulin

We next focused on these three compounds and evaluated at the molecular level whether they targeted plant tubulin but not mammalian tubulin. We thus compared their effect, at concentrations ranging from 0.1 to 50 µM on the in vitro assembly of tubulin purified from soybean or bovine brain. In contrast to colchicine and nocodazole, CM571, and CM852 even at high concentration (50 µM) did not show any significant effect on the assembly kinetics of bovine brain tubulin, in agreement with the lack of detectable changes in the cellular microtubule network of intact HeLa cells (Figure 4A). Of note CM094 induced a slight depolymerizing effect at 50 µM but had no effect at lower concentrations [27] While soybean tubulin was less assembly competent, we observed tubulin polymerization reaction under control conditions that was no longer detected in presence of dinitroaniline (Figure 4B). All three compounds impaired soybean tubulin assembly (Figure 4C-E). After dose-response analysis [27], the drug inhibitory profile is estimated as follows: dinitroaniline > CM852 > CM571 > CM094.

The comparison of the chemical structure of the compounds (Figure 5) showed that the three compounds belong to different classes of chemicals. No biological effects of CM571 have so far been described. Interestingly, CM094 shares common features with dinitroaniline, namely a nitro-phenylsulfonamide moiety, which may mediate the observed effect on plant tubulin assembly. Likewise, CM852 is a pyridasinone (n-chloridazon) that is currently used as a selective systemic herbicide [28]. Among other properties such as DNA intercalation and interference with the synthesis of fatty acids [29], n-chloridazon is primarily known to inhibit photosynthesis and thus to induce chlorosis, a symptom we observed in A. thaliana plantlets.

We concluded from these experiments that CM852, CM571, and CM094, originally selected by a virtual screen to bind to the dinitroaniline binding site of P. falciparum tubulin, were indeed able to target plant tubulin with no detectable effect on mammalian tubulin.

Selected Compounds Target Plant but not Mammalian Tubulin

We next focused on these three compounds and evaluated at the molecular level whether they targeted plant tubulin but not mammalian tubulin. We thus compared their effect, at concentrations ranging from 0.1 to 50 µM on the in vitro assembly of tubulin purified from soybean or bovine brain. In contrast to colchicine and nocodazole, CM571, and CM852 even at high concentration (50 µM) did not show any significant effect on the assembly kinetics of bovine brain tubulin, in agreement with the lack of detectable changes in the cellular microtubule network of intact HeLa cells (Figure 4A). Of note CM094 induced a slight depolymerizing effect at 50 µM but had no effect at lower concentrations [27] While soybean tubulin was less assembly competent, we observed tubulin polymerization reaction under control conditions that was no longer detected in presence of dinitroaniline (Figure 4B). All three compounds impaired soybean tubulin assembly (Figure 4C-E). After dose-response analysis [27], the drug inhibitory profile is estimated as follows: dinitroaniline > CM852 > CM571 > CM094.

The comparison of the chemical structure of the compounds (Figure 5) showed that the three compounds belong to different classes of chemicals. No biological effects of CM571 have so far been described. Interestingly, CM094 shares common features with dinitroaniline, namely a nitro-phenyl-sulfonamide moiety, which may mediate the observed effect on plant tubulin assembly. Likewise, CM852 is a pyridasinone (n-chloridazon) that is currently used as a selective systemic herbicide [28]. Among other properties such as DNA intercalation and interference with the synthesis of fatty acids [29], n-chloridazon is primarily known to inhibit photosynthesis and thus to induce chlorosis, a symptom we observed in A. thaliana plantlets.

We concluded from these experiments that CM852, CM571, and CM094, originally selected by a virtual screen to bind to the dinitroaniline binding site of P. falciparum tubulin, were indeed able to target plant tubulin with no detectable effect on mammalian tubulin.

Analysis of the Effects of the Compounds on Apicomplexan Parasites

Due to the sensitivity of T. gondii to dinitroanilines that has already been reported with IC50 values for commercially available dinitroanilines ranging from 45 nM to 6.7 µM, we tested whether the selected compounds could impact T. gondii and P. falciparum intracellular development within their respective host cells. Taking into account that the subpellicular MTs of extracellular parasites are non-dynamic and notoriously insensitive to MT pharmacological disruptors, we had to perform the assays on intracellular multiplying parasites. This experimental requirement implies that the compounds of interest could access not only the host cell cytosol, but also pass the membrane of the parasitophorous vacuoles, within which the tachyzoite multiplies and ultimately reach the parasite cytosol. Using fluorescent T. gondii tachyzoite and High Content Imaging that allowed quantitative detection of individual progeny within intracellular parasitophorous vacuoles post infection, we found, as expected, that the dinitroaniline analog oryzalin was highly and selectively potent on parasites, with an IC50 of 0.8 µM (Figure 6A, blue line). Oryzalin IC50 reached 99 µM for the human host fibroblasts (HFF). Accordingly the selectivity index, defined as the ratio of IC50 human fibroblasts/ IC50 T. gondii, reached 123.7. We found that compounds CM094 and CM571 moderately impacted parasite growth with an IC50 of 35 and 130 µM, respectively (Figure 6A, red and green lines), whereas compound CM852 had no detectable effect (Figure 6A, purple line). CM094 and CM571 showed no significant effect on human fibroblasts, with IC50 of 240 and 237 µM, respectively [30], resulting, however, in lower selectivity indexes than oryzalin (i.e. 6.8 and 1.8, respectively). The compound effectiveness towards its target, the dynamic MTs, is significantly challenged by the need of successive translocations across three biochemically different membranes. Thus, we next tested for a longer time period the activity of the most potent compound on T. gondii tachyzoite proliferation, defined with the IC50, i.e. CM094. To this end, we incubated 200 invasive tachyzoites on HFF monolayer in a six-well plate (9.61cm 2 /well) and added the drug immediately after one hour of invasion. The time window was adjusted to allow a single parasite to multiply and the progeny to undergo several rounds of infection or "lytic cycles". Under these conditions, we observed a significant difference in the size of HFF cleared areas between cells exposed to CM094 or to the vehicle alone. The effect was already observed after four days and drastically increased with time (Figure 6B,C). Assuming a conserved rate of infection between each sample, these assays document a significant loss of parasite fitness in presence of 15 µM of the compound and attest to the integrity of the HFF monolayer under drug treatment.

Analysis of the Effects of the Compounds on Apicomplexan Parasites

Due to the sensitivity of T. gondii to dinitroanilines that has already been reported with IC50 values for commercially available dinitroanilines ranging from 45 nM to 6.7 µM, we tested whether the selected compounds could impact T. gondii and P. falciparum intracellular development within their respective host cells. Taking into account that the subpellicular MTs of extracellular parasites are non-dynamic and notoriously insensitive to MT pharmacological disruptors, we had to perform the assays on intracellular multiplying parasites. This experimental requirement implies that the compounds of interest could access not only the host cell cytosol, but also pass the membrane of the parasitophorous vacuoles, within which the tachyzoite multiplies and ultimately reach the parasite cytosol. Using fluorescent T. gondii tachyzoite and High Content Imaging that allowed quantitative detection of individual progeny within intracellular parasitophorous vacuoles post infection, we found, as expected, that the dinitroaniline analog oryzalin was highly and selectively potent on parasites, with an IC 50 of 0.8 µM (Figure 6A, blue line). Oryzalin IC 50 reached 99 µM for the human host fibroblasts (HFF). Accordingly the selectivity index, defined as the ratio of IC 50 human fibroblasts/ IC 50 T. gondii, reached 123.7. We found that compounds CM094 and CM571 moderately impacted parasite growth with an IC50 of 35 and 130 µM, respectively (Figure 6A, red and green lines), whereas compound CM852 had no detectable effect (Figure 6A, purple line). CM094 and CM571 showed no significant effect on human fibroblasts, with IC50 of 240 and 237 µM, respectively [30], resulting, however, in lower selectivity indexes than oryzalin (i.e., 6.8 and 1.8, respectively). The compound effectiveness towards its target, the dynamic MTs, is significantly challenged by the need of successive translocations across three biochemically different membranes. Thus, we next tested for a longer time period the activity of the most potent compound on T. gondii tachyzoite proliferation, defined with the IC 50 , i.e., CM094. To this end, we incubated 200 invasive tachyzoites on HFF monolayer in a six-well plate (9.61 cm 2 /well) and added the drug immediately after one hour of invasion. The time window was adjusted to allow a single parasite to multiply and the progeny to undergo several rounds of infection or "lytic cycles". Under these conditions, we observed a significant difference in the size of HFF cleared areas between cells exposed to CM094 or to the vehicle alone. The effect was already observed after four days and drastically increased with time (Figure 6B,C). Assuming a conserved rate of infection between each sample, these assays document a significant loss of parasite fitness in presence of 15 µM of the compound and attest to the integrity of the HFF monolayer under drug treatment. The effect of selected compounds on P. falciparum intra-erythrocytic growth was tested. The growth of parasites within human red blood cells is determined using a classical SYBR Green assay followed by quantification of parasite DNA fluorescence. The graph presents growth curves for concentrations of each compounds ranging from 0 to 250 µM, in triplicates.

We then decided to investigate whether the compounds had an effect on the proliferation of P. falciparum (the most lethal agent of human malaria) during the intra-erythrocytic life stages, which are the symptomatic stages of the malaria infection. We thus incubated a tightly synchronized 3D7 ring wild-type population (i.e., initial intracellular developmental stage of the blood phase, 0.5% parasitaemia) in a 96-well plate (2% hematocrit) in the presence of different concentrations of the compounds ranging from 4 nM to 250 µM. Parasites were left in culture to undergo the intraerythrocytic life cycle of 48h, which usually allows the 3D7 strain to increase its population of about 10-fold. Parasitaemia after drug treatment was determined by a typical SYBR Green assay [31]. Compound CM852 (Figure 6D, purple line) showed highly variable effects on the parasite growth whereas both compounds CM094 (Figure 6D, red line) and CM571 (Figure 6D, green line) slowed down the intracellular proliferation of P. falciparum at high concentrations. The IC50 values of CM094 and CM571 were determined to be 94.7 and 122.4 µM, respectively. Notably, no cytotoxic effect could be observed on the human host erythrocytes [32], thus suggesting a specific effect on the parasite itself. The effect of selected compounds on P. falciparum intra-erythrocytic growth was tested. The growth of parasites within human red blood cells is determined using a classical SYBR Green assay followed by quantification of parasite DNA fluorescence. The graph presents growth curves for concentrations of each compounds ranging from 0 to 250 µM, in triplicates.

We then decided to investigate whether the compounds had an effect on the proliferation of P. falciparum (the most lethal agent of human malaria) during the intra-erythrocytic life stages, which are the symptomatic stages of the malaria infection. We thus incubated a tightly synchronized 3D7 ring wild-type population (i.e., initial intracellular developmental stage of the blood phase, 0.5% parasitaemia) in a 96-well plate (2% hematocrit) in the presence of different concentrations of the compounds ranging from 4 nM to 250 µM. Parasites were left in culture to undergo the intra-erythrocytic life cycle of 48h, which usually allows the 3D7 strain to increase its population of about 10-fold. Parasitaemia after drug treatment was determined by a typical SYBR Green assay [31]. Compound CM852 (Figure 6D, purple line) showed highly variable effects on the parasite growth whereas both compounds CM094 (Figure 6D, red line) and CM571 (Figure 6D, green line) slowed down the intracellular proliferation of P. falciparum at high concentrations. The IC 50 values of CM094 and CM571 were determined to be 94.7 and 122.4 µM, respectively. Notably, no cytotoxic effect could be observed on the human host erythrocytes [32], thus suggesting a specific effect on the parasite itself.

These results identified CM094, the compound most closely related to dinitroanilines as the only one to significantly impact the T. gondii tachyzoite lytic cycle. Both CM094 and CM571 were found to impact the trophozoite intra-erythrocytic development in P. falciparum.

Discussion

By integrating in silico and assay-based approaches we were able to explore the diversity of chemical space to find agents able to bind to the dinitroaniline tubulin-binding site. These compounds were assayed on evolutionary close and distant organisms, which allowed the identification of drugs with new chemical scaffolds that target plants and to a moderate extent, Apicomplexa parasites, while sparing animal cells.

Eleven compounds, out of the 82 in vitro-assayed compounds, were found active on the plant BY-2 microtubule cytoskeleton. This unusual high ratio points out the efficiency of the docking step strategy likely resulting from the combination of the following factors. First, a great number of ligands (300,000) have been docked thanks to the grid computational power, increasing the probability to select active compounds. Second, the S4MPLE algorithm and the access to the grid allowed an exploration of the possible conformations of the targeted site. Using a pre-docking step with known active and putative inactive ligands allowed the selection of the five most probable conformations. The docking on these five conformations was thus conducted on judiciously restricted but flexible targets. Finally, the docking poses and the important amount of interaction data have been scrutinized both manually and with homemade scripts. This in-depth analysis allowed the definition of a subset of criteria that were subsequently proven to be relevant.

While numerous compounds targeting mammalian tubulin are described, much less is known regarding drugs acting on plant tubulin. One reason is that the systematic research of compounds that selectively affect plant tubulin assembly is hampered by the poor availability of purified assembly competent plant tubulin. High content screening methods, based on the visualization of the compound effect on plant cell microtubule networks, could represent a convenient alternative. Here, we did not use computer-based methods for the analysis of the high content screening experiments, which allowed the evaluation of compound effects on BY-2 cell microtubules, but care has been taken to develop a standardized protocol that could be readily automated. BY-2 cells have proven to be a good cell model for this screening but other plant cells could also be valuably used in similar screens. We anticipate that high throughput screens of the effect of large sets of compounds on plant proteins in the cellular context will be easily implemented and benefit of our methodological developments. Such a method could also be used in a chemical genetics approach to identify chemically targetable microtubule regulators [33,34].

Biochemical experiments confirmed that plant tubulin is the in vitro target of the three selected compounds and that their binding to tubulin is likely responsible for MT depolymerization. Their toxic effect on plant cells and their effect on plant growth can therefore most probably be attributed to their effect on tubulin dynamics. It has been shown, however, that oryzalin also induces changes in the morphology of the endoplasmic reticulum (ER) and Golgi apparatus, which could contribute to its herbicide properties [35]. Such additional effects could also be responsible of the observed herbicide activity of the compounds we have selected, especially CM094, which shares some structural similarities with dinitroaniline.

Interestingly compound CM852 (pyrazon [5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone]) is already described as an herbicide that works by blocking electron transport in photosystem II in green plants, thereby inhibiting photosynthesis. Its inhibitory effect on tubulin assembly, uncovered in this work, is likely reinforcing its toxic effect on plants. Available toxicity data on this compound indicate that it is of low toxicity without highly specific responses in mammals. This study confirms the selectivity of CM852 and the other two selected compounds for plant cells since none of them affected human fibroblastic and epithelial cell viability, in full consistency with the fact that they do not target mammalian tubulin. Moreover, the absence of their toxicity indicates that these compounds are not highly reactive and that they do not target proteins important for cell viability. Notably, since at least the CM094 compound has proven some activity on T. gondii intracellular growth and to a lesser extent on P. falciparum. This argues that CM094 can cross multiple biological barriers, and remain active. Remarkably, to target MTs dynamics that occur during apicomplexan zoites multiplication (i.e., T. gondii tachyzoites and P. falciparum trophozoite), the drug has to successfully translocate across the host cell plasma membrane, the membrane of the vacuole which houses the replicating tachyzoites and the plasma membrane of the tachyzoite itself. Whether the anti-tachyzoite MT activity of CM094 is reduced due the multistep obligate trafficking of the drug to reach its target once delivered into the culture medium remains to be evaluated. Previous work on the effect of dinitroanilines on P. falciparum corroborate our results showing a moderate activity on P. falciparum, but a very low mammalian cytotoxicity of the compounds [36]. This work also concluded that due to their hydrophobic nature, dinitroaniline derivatives largely accumulate in the parasite membranes. This reduces the amount of molecules having access to their microtubular target, likely explaining the modest effect observed on P. falciparum. Synthesis of chemical derivatives of CM094 may overcome this limitation and gain in anti-malarial activity.

The development of new herbicides, with no negative impact on humans and on the wild fauna, would have important consequences at the ecological and economical levels. While the compounds we have described act in the micromolar range, their chemical structure is simple. These molecules thus provide a useful platform for compound optimization. They also exhibit a selective effect on T. gondii and P. falciparum proliferation and could thus represent alternative scaffolds to the dinitroaniline analogs, in particular the meta-amino derivatives that have been already characterized as potent anti-T. gondii reagents [37]. Considering the urgent need for the development of therapeutic agents against malaria and other parasitic diseases, uncovering new scaffolds as leads for the future design of selective anti-parasitic drugs remains a priority.

Materials and Methods

Chemical Reagents, Recombinant and Purified Protein

Reagents used include DMSO (Sigma, D5879, Saint-Quentin Fallavier, France); oryzalin (Sigma, 36182); paclitaxel (Sigma, T1912); colchicine (Sigma, C9754); nocodazole (Sigma, M1404); phosphate buffer saline (PBS, Sigma P4417); foetal bovine serum (FBS, Sigma); formaldehyde (Sigma, F1635); glutaraldehyde (Polysciences, Inc, Warrington, PA, USA); Tween 20 (Sigma, P9416); sodium azide (Merck, Lyon, France, 6688); piperazine-N,N -bis(2-ethanesulfonic acid (PIPES, Sigma, P6757); ethylene glycol-bis(β-aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA, Sigma, E4378); MgCl 2 (Sigma, M1028); Triton X100 (Sigma, T8787); Glycerol (Sigma); phenylmethylsulfonyl fluoride (PMSF, Sigma); 2-(N-morpholino)ethanesulfonic acid (MES, Sigma, M3671); 4 ,6-diamidino-2-phénylindole (DAPI, Sigma, D8417); CaCl 2 (Sigma, C5670); mannitol (Sigma, M4125); pectolyase Y23 (Seishin Pharmaceutical, Tokyo, Japan); macerozyme R-10 (Serva, Heidelberg, Germany); caylase 345 (Cayla, Toulouse, France); Hoechst 33342 reagent (Sigma, B2261); protease cocktail inhibitors (Sigma, P8340); phosphatase cocktail inhibitors (Sigma, P5726); bovine serum albumin (BSA, Sigma, A3059); normal goat serum (Interchim, Montluçon, France); 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT); Thiazolyl blue (Sigma, M5655). Tubulin was prepared from fresh bovine brains as described in Paturle-Lafanechère et al. [38]. Soybean tubulin was purchased from Cytoskeleton, Inc. (Denver, CO, USA). Compounds screened were from ChemBridge Corporation (San Diego, CA, USA).

Antibodies

The α-tubulin antibody used was from clone α3a [39]. Anti-mouse IgG secondary antibodies conjugated with cyanine 3 were from Jackson ImmunoResearch Laboratories (Cambridgeshire, UK).

Mammalian, Plant and Apicomplexan Cell Lines

Mammalian cells: HeLa cells were originally purchased from the American Type Culture Collection (ATCC, Middlesex, UK), and maintained in Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin, instead of DMEM, because the assayed compounds were more soluble in RPMI. MES-SA cells and MES-SA-DX5, from ATCC, were first grown in McCoy's 5A (ATCC) with 10% (v/v) FBS and 1% penicillin/streptomycin and further adapted to grow in RPMI-1640 with 10% (v/v) FBS and antibiotics. These mammalian cell lines were maintained at 37 • C with 5% CO 2 and 3% O 2 . Cells were treated with the compounds for 2 h unless otherwise stated. Plant cells: Tobacco BY-2 cells (Nicotiana tabacum L. Bright-Yellow 2, Riken) were grown in suspension according to Nagata et al. [40]. Primary human foreskin fibroblasts (ATCC CRL-1634) were seeded at about 70-80% of confluence in P96 and P6 well plates in high glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 10 mM HEPES pH 7.0 (i.e. complete medium) and used 48-72 h later when confluent. Apicomplexan cell lines: The T. gondii RH strain expressing cytosolic GFP was maintained by serial passage in Human Foreskin Fibroblats (HFF) monolayers maintained in complete medium and as previously described [41]. P. falciparum 3D7 cultures were grown, as previously described [42] in 2% hematocrit (obtained from Etablissement du Sang Français, Grenoble, France) in RPMI medium (Thermo Fisher Scientific, Waltham, MA, USA) complemented with 10% Albumax (Thermo Fisher Scientific), Gentamycin (Merck Sigma Aldrich, Lyon, France) and hypoxanthine at 37 • C with a beta gas mix (1% O 2 , 5% CO 2 , 96% N 2 ). The dinitroaniline binding site of α-tubulin has been described in previous studies, located beneath the H1-S2 loop [13]. A sub-library of compounds, comprising 307,802 molecules available at ChemBridge Corporation was retrieved from the ZINC database (http://zinc15.docking.org/) in mol2 format with the hydrogen atoms and the atomic partial charges. The virtual library was massively docked on the EGEE (Enabling Grids for E-sciencE) European grid infrastructure [43] with the FlexX software [44] at the level of the putative dinitroaniline/oryzalin site. FlexX docking results contained several groups of data: (1) for each compound docked on each protein conformation, the 10 best poses were returned in mol2-format output files; (2) associated to each pose, the software returned also files giving the FlexX score value and the interactions between the protein residues and the ligand. The amount of information associated with 10 poses of 307,802 molecules docked on 7 active sites represented about 300 GB of textual data. To process this huge amount of data, analysis scripts were developed in Unix Shell, Unix Awk, or Perl languages; in order to visualize and sort the interesting values, the R programming language and the Kyplot package were used for statistics. To visualize, align ligands and to inspect, manually, their interactions with the dinitroaniline/oryzalin active site, the following software were used: Visual molecular dynamics VMD [45] Chimera [46], Ligplot [47]), MOE (Molecular Operating Environment, Chemical Computing Group-1010 Sherbrooke Street West, Montreal, Canada H3A2R7) and Hermes, the graphical interface of Gold [48].

Microtubule-Interference Assay in BY-2 Tobacco Cells

Samples of 90 µL of BY-2 cells, grown for 2.5 to 6.5 days, were transferred into 96-well polystyrene plates (Masterblock 2 mL, Greiner, Courtaboeuf, France) and supplemented with 10 µL of the chemical compounds (final concentration 50 µM). The cells were then incubated for 2 h at 27 • C in the dark and under gentle agitation. Cells were further fixed by the addition in each well of 900 µL of MBS buffer (50 mM PIPES, 5 mM EGTA, 1 mM MgCl 2 , 2% Glycerol, pH 6.9) containing 3.7% formaldehyde, 1% glutaraldehyde, 1% DMSO, 0.5% Triton X-100 and 200 µM phenylmethane sulfonyl fluoride (PMSF), for 15 min under gentle agitation. The fixation buffer was then removed after sedimentation of the cells under centrifugation (2 min, 100× g) and replaced by 500 µL of MBS buffer for 10 min. This washing step was repeated 4 times. A volume of 50 µL of cell suspension was then transferred from Masterblock plates to Microclear 96-well flat bottom polypropylene plates (Greiner Bio-One #655090) coated with poly-D-Lysine. After a 15 min sedimentation period, cell walls were permeabilized for 2 min with 25 mM MES, 8 mM CaCl 2 , 600 mM Mannitol, 0.02% Pectolyase, 0.1% Macerozyme, 0.3% Caylase at pH 5.6, at room temperature. They were then washed with 50 µL of MBS buffer, and incubated with 5% of normal goat serum for 2 h. After 5 washes with MBS buffer, 45 µL of anti-tubulin antibodies at 1/5000 dilution were added in each well and incubated overnight. After 5 washes with MBS buffer, cells were incubated with Cy3 secondary antibodies (dilution 1/2000) and Hoechst (dilution 1/1000) for nuclei staining for 2 h. Finally, after 5 washes in PBS, the BY-2 cells were stocked in 50 µL of PBS/glycerol (50/50) at 4 • C before automated imaging. Compounds found to be active were systematically tested again from freshly made solutions.

Microtubule-Interference Assay in Hela Cells

The effect of compounds on microtubules was assayed in HeLa cells in 96 well-microplates using immunofluorescence, after permeabilization, and fixation of the cells, as previously described [16].

Drug Effect on Toxoplasma gondii and Plasmodium falciparum Parasite In Vitro

The activity of compounds against T. gondii in vitro was tested by (i) automatic scoring of GFP-expressing parasites within intracellular vacuoles over 30 h post invasion and in presence of a wide range of compound concentration. These assays allowed assessing the growth potential over about 4 to 5 replication cycles (ii) analysis of clear zones developing in a host fibroblast monolayer over 4 and 7 days as a result of the cell lysis induced by tachyzoite multiplication following infection by a single parasite. The so-called comparative plaque assay therefore revealed the additive defects on growth, motile, invasive and egress capabilities caused by the compound under study over numerous cycles of infection. To first monitor parasite multiplication over the 30 h period, we applied high content screening using fluorescence microscopy on 96 well plates with 20 fields of acquisition for each well and performed in triplicates for each condition. Images were further processed with ScanˆR software (Olympus Life Science, Rungis, France). In the plaque assay, area of clear zones in the HFF monolayer were defined by image segmentation and measured following ethanol fixation and Crystal violet (0.05% in distilled water) staining with ImageJ software (https://imagej.nih.gov/ij/, [49]) and for all assays the datasets were analyzed using GraphPad Prism 6 software (La Jolla, CA, USA) and organized using Adobe Photoshop CS6 software (Paris, France).

Activity of compounds on P. falciparum intra-erythocytic life stages was assayed on tightly synchronized cultures of 3D7 ring culture using a classical SYBR Green assay, as previously described [31]. Briefly, populations of infected red blood cells with 0.5% 3D7 P. falciparum rings were allowed to grow in culture medium containing various concentrations of compounds (ranging from 4 nM to 250 µM) for an entire intra-erythrocytic life cycle of 48 h in 96-well plates (Thermo Fischer Scientific). Parasites (i.e., infected red blood cells) were quantified using SYBR Green II (Merck Sigma Aldrich), which gives a green fluorescence upon its contact with P. falciparum DNA (erythrocyte lacking nuclei and DNA, fluorescence is specific to the parasite presence). Fluorescence quantification was measured using a CLARIOstar 96-well-plate reader (BMG Labtech, Champigny s/Marne, France) and data was analyzed using GraphPad Prism 6 software.

Automated Imaging

Automated imaging of cells seeded in 96-well clear-bottom plate (Greiner, #655090) was performed on INCell Analyzer 1000 (GE-Healthcare, Cardiff, UK) using an 20x air objective. Eight images/well were acquired in the center of the well. Excitation and emission filters pairs used for nuclei, A488 and Cy3 staining were 360 nm/460 nm, 475 nm/535 nm and 535 nm/620 nm, respectively. Acquisition parameters such as exposure time and Z-plane focus were specifically fixed for each immunofluorescence assay.

1 ACKNOWLEDGEMENTForemost

 1 , I would like to express my deepest gratitude to my thesis supervisors Dr. Cyrille Botté and Dr. Yoshiki Yamaryo Botté for their full support, expert guidance, understanding and encouragement throughout the course of this study. I am extremely grateful to both for mentoring me to my thesis completion. I thank Yoshiki for her timely counsel and an eye for detail that trained me rigorously during my PhD and Cyrille for allowing me to work with new ideas and for always having his office door open in case of doubts and discussions. I would like to thank team Apicolipid-Cyrine, Christophe, Serena, Samuel and Nick for making the work environment cheerful. A special thanks to Annie, my first tutor of Plasmodium molecular biology, for the fun times and constant support; and Nick, ParaFrap partner, for being ever ready to help and for answering my unending scientific queries. I acknowledge ParaFrap for their financial support and coursework during my thesis. I would also like to thank my thesis committee members Dr. Ali Hakimi and Dr. Jose Juan Lopez Rubio and ParaFrap scientific advisory board. Their thoughtful questions and comments are valued greatly. I also would like to express my gratitude to my thesis jury members Prof. Malcolm McConville, Prof. Dominique Soldati, Dr. James Macrae and Prof. Pierre Hainaut.

Fig. 1 T

 1 Fig. 1 T. gondii LIPIN (TgLIPIN) is a phosphatidate phosphatase localized to parasite cytoplasm ....................................................................................................................... Fig. 2 TgLIPIN is indispensable for parasite replication and growth within its host . Fig.3 TgLIPIN depletion results in gross membrane anomalies early on during the process of ATc downregulation ...................................................................................... Fig. 4 TgLIPIN regulates critical levels of PA and other major phospholipids ............ Fig. 5 TgLIPIN generated DAG is directed towards neutral lipid storage .................. Fig. 6. Monitoring the source of excess fatty acids in TgLIPIN-iKD ............................ Fig. 7 Proposed role of LIPIN in Toxoplasma lipid metabolism ...................................

Figure 2

 2 Figure 2 Complete life cycle of Plasmodium falciparum with asexual development in human host cells, hepatocytes and erythrocytes (a), adapted from[START_REF] Cowman | Malaria: Biology and Disease[END_REF]) and sexual development involving life stages female/male gametes, ookinetes, oocysts and sporozoites within female Anopheles mosquito (b), adapted from(Aly, Vaughan, and Kappe 2009). The initiation of asexual life cycle within human host is initiated by the bite of Anopheles injecting sporozoites into the dermis. This is followed by their transport to liver known as the pre-erythrocytic stage where they divide by schizogony (over a period of 10 days) to generate tens of thousands of daughter merozoites are released in packets of merosomes into the bloodstream. Within the bloodstream, these merozoites invade red blood cells carrying out their erythrocytic life stage, a cycle of which lasts for over 48 h. Next, a 15 days period leading to intra-erythrocytic gametocyte development follows which enters the peripheral circulation for ingestion by a mosquito for further transmission. The first part of sexual life cycle occurs in the mosquito midgut where the gametocytes emerge as male and female gametes followed by their fusion into a zygote which transforms into ookinete over a period of 24 h. the ookinete encysts to become oocyst where following sporogony over a period of 10-14 days, thousands of sporozoites are produced. These sporozoites traverse to enter into mosquito salivary gland from where they are transmitted to mammalian host over next blood meal.

Figure 3 .

 3 Figure 3. a) Infection of T. gondii within the intermediate host (asexual cycle) occurs through ingestion of contaminated food. The transmissible bradyzoites or sporozoites are released into the intestinal lumen where they invade intestinal enterocytes and differentiate into tachyzoites. Tachyzoites divide rapidly by the process of endodyogeny (b) and then egress from the host, infecting neighboring cells to start another cycle. Tachyzoites convert to dormant stage bradyzoites and then finally to tissue cysts that remain persistent within brain or musculature for almost lifetime of the intermediate host(a).The sexual cycle occurs only in cats, initiated by rupture of ingested tissue cyst that releases bradyzoites into enterocytes. These bradyzoites then undergo a self-limiting number of asexual multiplications, characterized by the development of merozoites within schizont, a process called schizogony (c). This is followed by gamogony and fertilization of the gametes forming an unsporulated oocyst (d). These are releases by with cat feces into the external environment where they undergo sporulation generating at least 4 haploid sporozoites per oocyst (e). (a) adapted from[START_REF] Pittman | Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections[END_REF]; (b,c) adapted from(Francia and Striepen, 2014)

Figure 4

 4 Figure 4 Schematic representation of active invasion process of T. gondii inside its host

Figure 5 a

 5 Figure 5 a) Schematic enlarged view of the apical complex cytoskeleton, showing the conoid (green),preconoidal, and polar rings (brown), and two intraconoid MT (green) (adapted from[START_REF] Hu | Cytoskeletal components of an invasion machine -The apical complex of Toxoplasma gondii[END_REF], b) TEM image of the apical complex of Toxoplasma tachyzoite (adapted from[START_REF] Dubey | Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts[END_REF]

Figure 7 Figure 8 .

 78 Figure 7 Metabolic pathways present in apicoplast (a) (adapted from[START_REF] Ralph | Metabolic maps and functions of the Plasmodium falciparum apicoplast[END_REF] and mitochondria (b) (adapted from[START_REF] Oppenheim | BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei[END_REF]. Apicoplast provides essential metabolites for parasite's intracellular survival: fatty acids via FASII pathway, isoprenoid biosynthesis (IPP), iron-sulfur cluster biosynthesis and haem synthesis. Mitochondria resident tricarboxylic acid cycle contributes to parasite's ATP and anabolic needs

Figure 10

 10 Figure 10 Phosphatidic acid biosynthetic pathways in T. gondii. a) Apicoplast-localized pathway of PA generation involving acyltransferases TgATS1 and TgATS2 (adapted fromAmiar et al., 2016Amiar et al., , 2019)). b) Generation of signaling species of PA at the parasite plasma membrane and vacuolar space by the action of diacylglycerol kinases TgDGK1 and TgDGK2 (adapted fromBullen et al., 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF]. Other abbreviations used, PAP, phosphatidic acid phosphatase; PKG, protein kinase G; PI-PLC (adapted from Fang,Marchesini and Moreno, 2006);phosphoinositide phospholipase C; PI, phosphatidylinositol; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol.

Figure 11

 11 Figure 11 Schematic representation of various known functions of phosphatidic acid (PA) in apicomplexa. PA acts as the central precursor molecule aiding in generation of membrane phospholipids (a)(Amiar et al., 2016) and cell division process by maintaining a balance with its own precursor (LPA)(Amiar et al., 2019) (b). This dynamic lipid also acts as signaling molecule participating in keys parasite pathogenicity events like invasion and egress(Bullen et al., 2016;[START_REF] Bisio | Phosphatidic Acid Governs Natural Egress in Toxoplasma Gondii via a Guanylate Cyclase Receptor Platform[END_REF]. PA is also channeled towards lipid storage pathway ([START_REF] Nolan | Novel Approaches to Kill Toxoplasma Gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite[END_REF] this thesis).

Figure 9

 9 Figure 9 Schematic representation of overall phospholipid biosynthetic pathways within eukaryotes. Enzymes outlines with dark green and red have been characterized in T. gondii and P. falciparum, respectively. Abbreviations used: CK, choline kinase; CCT, choline phosphate: cytidyltransferase; CPT, diacylglycerol choline phosphotransferase; PTS, phosphatidylthreonine synthase; PMT, phosphatidylethanolamine methyltransferase; SD, serine decarboxylase; EK, ethanolamine kinase; ECT, ethanolamine phosphate: cytidyltransferase; EPT, diacylglycerol ethanolamine phosphotransferase; PSS, phosphatidylserine synthase; PIS, phosphatidylinositol synthase; PGPS, CDP-DAG-glycerol-3-phosphate-3-phosphatidytransferase; PGPP, Phosphatidylglycerophosphatase; CLS, cardiolipin synthase; CDS, cytidine dinucleotide-diacylglycerol synthase; AGPAT, 1-acylglycerol-3-phosphate-o-acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; DGAT, acyl-CoA: diacylglycerol acyltransferase

Figure 12

 12 Figure 12Representation of fatty acid salvage and biosynthetic pathways in Toxoplasma. The parasite lipids are a patchwork of fatty acids derived from the host and de novo biosynthesis. T. gondii can synthesize fatty acid de novo via FASII (a) and elongate their chain lengths via elongation machinery (b). Apicoplast-localized FASII produces myristic (C14:0), palmitic acid (C16:0) in addition to lipoic acid (a). b) ER-associated elongase system synthesizes long chain monounsaturated fatty acids using the activity of elongases (ELO), ketoacyl-CoA reductase (KCR), hydroxyacyl-CoA dehydratase (DEH) and enoyl-CoA reductase enzymes (ECR) from substrates derived from FASII and acetate metabolism (TgACS, acetyl CoA synthetase; TgACC2, acetyl CoA carboxylase 2). c) T.gondii is also able to scavenge fatty acids directly from the host, including C14:0, C16:0, C16:1, C18:2, C20:4. Other abbreviations used: TgACC1, acetyl CoA carboxylase 1; ACP, acyl carrier protein; PDH, pyruvate dehydrogenase; PK, pyruvate kinase; TPT, triose phosphate transporter; PEP, phosphoenol pyruvate (adapted from[START_REF] Ramakrishnan | The intracellular parasite Toxoplasma gondiidepends on the synthesis of longchain and very long-chain unsaturated fatty acids not supplied by the host cell[END_REF].

Figure13

  Figure13 Schematic representation of known methods by which parasite scavenge fatty acids from the host. a),b) TEM showing entrapped host derived lipid droplets (hLDs) into the intravacuolar tubular network within the parasitophorous vacuole (a) (adapted from Nolan, Romano and Coppens, 2017)and recruitment of host organelles, mitochondria (m), endoplasmic reticulum (ER) (b) towards the parasitophorous vacuolar membrane (PVM) (adapted from Coppens et al., 2006). c) IFA image localizations of various ATP-binding cassette transporters, TgACBG107, TgABCG89, TgABCG77 ,TgABCG87 .TgABCG87 ,TgABCG84 involved in import and export of specific lipid between host and parasite (adapted from Ehrenman et al., 2010). d) IFA showing mitochondrial (TgACBP2, (adapted from[START_REF] Fu | Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism[END_REF] and cytoplasmic localizations (TgACBP1, TgSCP1, (adapted from[START_REF] Fu | Synergistic Roles of Acyl-CoA Binding Protein (ACBP1) and Sterol Carrier Protein 2 (SCP2) in Toxoplasma Lipid Metabolism[END_REF] of FA transporters present within the parasite. e) Overview of T. gondii secretory endocytic pathway also assisting in the incorporation of lipids from the external environment (5) (adapted from[START_REF] Gras | An endocytic-secretory cycle participates in Toxoplasma gondii in motility[END_REF]. f) PVM allows passage of small molecules including lipids, with the help of dense granule proteins TgGRA17 and TgGRA23 (adapted from[START_REF] Gold | The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole[END_REF].

  Fu, Y. et al. (2019) 'Synergistic roles of acyl-CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in HYPOTHESIS AND AIMS OF THE THESIS Lipid metabolism in apicomplexan parasites has been proven to be essential for the generation of infectious progeny and persistence within their hosts. The production of fatty acids (FA) is central to parasite lipid metabolism as they constitute essential hydrophobic building blocks required for membrane lipid synthesis. Apicomplexa was long thought to suffice their fatty acid needs solely by scavenging from the host cell but this hypothesis was challenged by the discovery of a prokaryotic fatty acid synthesis pathway: FASII within a non-photosynthetic plastid called apicoplast. Thus, we propose that parasite lipids are a patchwork of fatty acids synthesized de novo and/or scavenged from the host. These parasites infect various kinds of hosts and therefore encounter different nutritional challenges/host environments. Recent work (including ours) in the field of apicomplexa biology showed that the lipid synthesis and intracellular parasite development are also dependent on the host environment/nutritional conditions[START_REF] Blume | Metabolic interactions between Toxoplasma gondii and its host[END_REF] Amiar et al. 2019). However, the molecular mechanism by which these parasites combine and regulate the fatty acid flux from these two sources in context of the host nutritional status to sustain their intracellular survival, remains unknown.My PhD project focused on understanding how and why the parasite utilizes both FA sources to form a 'patchwork lipid'. In quest for the same, I investigated two important metabolic pathways and their enzymes: a) for the activation of the FA building blocks by acyl coA synthetases (ACS) and b) use of FA to generate central lipid precursor, phosphatidic acid (PA) and diacylglycerol (DAG) by phosphatidic acid phosphatase. We hypothesized that lipin and ACSs act at the crossroads of utilization of FA from the two sources-de novo machinery and scavenging from host for building of central precursor lipid molecules within the parasite.In pursuit of the same, my thesis projects aimed for the following objectives: i. Functional characterisation of lipin proteins in T. gondii and P. falciparum by generation of genetic knockouts and core lipidomics using GC-MS.ii. Identification of putative candidate genes encoding acyl-CoA synthetases in T. gondii and their subsequent functional characterisation by lipidomics to determine their expected roles as fatty acid activators allowing their further trafficking and utilisation by the parasite iii. Elucidating the role of host nutritional status on the parasites to ascertain adept metabolic capacity of the parasite. iv. Developing novel fluxomic approaches using different stable isotope substrates ( 13 C-U-Glucose,31 d-C16:0) to determine the different sources and flux of FAs from synthetic organelles and/or from host and its environment contributing to the formation of parasite lipids. CHAPTER IV: LIPIN, A PIVOTAL NEXUS IN TOXOPLASMA LIPID METABOLISM, CHANNELING HOST FATTY ACID FLUX TO STORAGE AND MEMBRANE BIOGENESIS CHAPTER IV SUMMARY Lipid biosynthesis and metabolism in apicomplexan parasites is reliant on an essential combination of fatty acid flux derived from de novo synthesis and scavenging from the host. The survival of these parasites within their hosts requires a balance between the different sources of FA flux directed towards lipid synthesis for generation of membranes, signaling events and storage. In this study we characterized a key phosphatidic acid phosphatase TgLIPIN participating in the parasite FA homeostasis by regulating the critical levels of central precursor lipid molecules phosphatidic acid (PA) and diacylglycerol (DAG).

  interactions Membranaires normales et pathologiques, UMR5235, Université Montpellier II, France.

  Apicomplexans are obligate intracellular parasites responsible for major human diseases worldwide. Their intracellular survival relies on intense lipid synthesis feeding membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identified an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveals that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using novel fluxomics approaches, we uncovered the first parasite host-scavenged lipidome and showed that TgLIPIN prevents parasite 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triglycerides and membrane phospholipids.

(

  PF3D7_0303200), P. berghei (PBANKA_040180) , Homo sapiens (NX_Q14693), H. sapiens (NX_Q92539), H. sapiens (NX_Q9BQK8), Saccharomyces cerevisiae _PAH1 (PAP1, SMP2, YMR165C, YM8520.14C), Cyrptosporidium parvum (cgd3_3210), Cyanidioschzyon merolae (CYME_CMN061C), Neospora caninum (BN1204), Hammondia hammondi (HHA_230690), Chlamydomonas reindhartii (CHLRE_12g506600v5), Arabiodopsis thaliana_AtPAH1 (At3g09560), Arabidopsis thaliana_AtPAH2 (At5g42870), Leishmania major (LMJF_06_0830),Trypanosoma brucei (Tb927.7.5450), Chromera velia (Cvel_24403). First step involved the curation of these protein sequences. The protein sequences were aligned (ClustalW) and then gaps were removed from the alignment. Finally, the phylogenetic tree was constructed using the maximum likelihood method in the PhyML program. The default substitution model (WAG) was selected. Graphical representation and edition of the phylogenetic tree were performed with Cladogram.

  , two separate homology flanks were chosen. The 5' flank was amplified 1637 bp upstream of the TgLIPIN start -3'. The pcr product was ligated to PacI and NdeI digested vector pPR2 using NEB assembly reaction. Next, the 3' flank was amplified as a 1739 bp fragment beginning at the start codon of TgLIPIN with reverse 5′-GCACTGACTGGCATGAATGGCCAGGCGCTGCCTTCTTTCCATTC-3'. The 3' homology flank was annealed to XmaI and NotI digested pPR-HA3-DHFR vector that already contained the TgLIPIN 5' flank. The final cloned vector positions the start codon of TgLIPIN downstream of the ATc-regulatable t7s4 promoter and a 3xHA tag. The resulting vector with NotI and transfected this into TATiΔku80 parasites. Parasites were selected with the drug pyrimethamine and cloned by limiting dilution. Screening of parasite clones where the t7s4 promoter had successfully replaced the native TgLIPIN promoter, was done using the primers P1 5'-CGATGACCTGTGTCGACCTGT-3' P2 5′-TCTTCTTTGAGGGAAGAGGAAACG-3', P3 5'-GGTACCGAGCTCGACTTTCAC-3', P4 5'-CAGCTGATCGGAGGTTGGTCT-3' and P5 5'-CTCCACCGTTTCCGGTTCCGT-3' in the combinations described in supplementary fig S2. All PCRs were performed with TaKara primestar max polymerase. The knockdown of TgLIPIN was induced with 0.5 μg ml -1 of anhydrotetracycline (ATc).

PfLIPIN

  was tagged at the C-terminus with 3×HA. The construct used for transfection, ptex-HAglmS, contained 867 bp of homology sequence (3' homology region) immediately upstream of the stop codon of PfLIPIN (Pf3D7_0303200). The 3' homology flank was PCR amplified from P. cloned into the BglII and PstI sites of ptex-HAglmS using neb assembly reaction mix(Counihan

FIGURESFig. 1 TFig. 2

 12 FIGURES

Fig. 3

 3 Fig.3 TgLIPIN depletion results in gross membrane anomalies early on during the process of ATc downregulation a) Transmission electron micrograph representing a normal parasite with regular nucleus and other endomembrane organelles. b) TEM image showing evaginated IMC (encircled and zoomed in image b'), in TgLIPIN-ikD + ATc (12 h). c) IFA representation of aberrant IMC biogenesis s in the TgLIPIN-ikD (24 h +ATc) parasites by probing with anti-IMC1 antibody (red). d) The nuclei in TgLIPIN-ikD (24 h +ATc) parasites appear multi-lobed characterized by membranous extensions into the cytoplasm, marked with red overlay on the electron micrograph. e) TgLIPIN-ikD (24 h + ATc): Intracellular replication defect represented as multiple daughter cells (DC) arrested within two parasite mother cells.

Fig. 4 Fig. 5 Fig. 6 .

 456 Fig. 4 TgLIPIN regulates critical levels of PA and other major phospholipids a) Representative glycerolipid biosynthesis pathway in eukaryotes. TgLIPIN catalyzes the biochemical conversion of PA to DAG. PA, can be channeled into major membrane phospholipids through CDP-DAG pathway. On the other hand, DAG generated by catalytic action of TgLIPIN is specifically channeled towards triglyceride biosynthesis (TAGs) b) Relative abundance of PA in TgLIPIN-iKD (24 h and 48 h +ATc) is represented as ratio of nmol/parasite no. in +ATc and -ATc. TgLIPIN depletion induces approx. 3-fold rise PA relative abundance in the +ATc vs -ATc conditions. c) Fatty acid composition of PA in TgLIPIN-ikD (+/-ATc) in Mol%. d) Relative abundance of DAG (+ATc/-ATc) is reduced almost to 0.5 by TgLIPIN depletion (24 h and 48 h + ATc).e) Relative phospholipid abundance (+ATc/-ATc) is increased by TgLIPIN depletion using +ATc for 24 and 48 h. Error bars = standard error of the mean, n=3. p values are indicated as follows: 0.01-0.05=*, 0.01-0.001=** and <0.001=***

Fig. 7

 7 Fig. 7 Proposed role of LIPIN in Toxoplasma lipid metabolism Schematic representation of the dual essential role of TgLIPIN in parasite lipid metabolism including, membrane biogenesis and lipid storage. a) The parasite utilizes fatty acids derived from both apicoplast FASII and host to synthesize major lipids, beginning with phosphatidic acid (PA). PA is hydrolyzed to diacylglycerol (DAG), which is further acylated to generate triacylglycerol (TAG). The other branch of this pathway redirects PA towards generation of major membrane phospholipids via CDP-DAG pathway. The FA homeostasis between membrane biogenesis and storage essential for normal parasite development within its host is maintained by the PA phosphatase TgLIPIN. (b) Genetic ablation of TgLIPIN in the presence of anhydrotetracycline (ATc) results in PA/DAG imbalance. The increased PA is channeled towards CDP-DAG pathway resulting in excess phospholipids and consequent gross membrane anomalies within the parasite IMC and nucleus. Simultaneously, the reduction in DAG affected TAG biogenesis and hence the lipid storage capacity, marked by decreased lipid droplets, within the parasite lacking TgLIPIN. The impairment of TAG biosynthesis, resulted in excess fatty acids derived from the host, within TgLIPIN mutant, causing lipotoxicity.

Fig.

  Fig. S2 a) Schematic of molecular strategy for generation of TgLIPIN-ikD tagged with 3*HA at its N-terminus. b) Confirmation of correct promoter replacement in the TgLIPIN-ikD line. Primers combinations during the screening PCR yielded products of expected amplicon size validating the mutant. The template genomic DNA was extracted from TgLIPIN-ikD line (M) and RH∆ Ku80 TATi strain (WT) (control). Primers 5+4 generated 1890 bp amplicon with RH∆ Ku80 TATi strain (WT) and approx. 4800 bp using TgLIPIN-ikD gDNA (M), suggesting incorporation of tetracycline inducible elements along with DHFR cassette at the genome locus of TgLIPIN. Expectedly, primers combinations specific to Tet-regulatable elements (1+2, 3+4) generated an amplicon approx. 3000 bp in size only using TgLIPIN (M) as the gDNA template.

  were identified to have highly conserved ACS motifs: Motif I-{Y,F} TSG {T,S} TGXPK; Motif II-TGDX(7) GX hX (2) RX (4) Hx(3,4) GX (2) hX (4) hE ; Motif III-YGXTE (WWQTE); Motif IV-LPLXH; Motif V-LPLSH, V-PKTX{S,T} GKIX {R,K} (KXX {R,K})(Watkins et al. 2007) (Fig.ure 1a,b). We then named the 7 putative T. gondii ACS (TgACS) candidates as TgACS1 (TGGT1_297220), TgACS2 (TGGT1_310150), TgACS3 (TGGT1_310080), TgACS4 (TGGT1_243800), TgACS5 (TGGT1_247760), TgACS6 (TGGT1_ 232580), TgACS7 (TGGT1_276155) (Fig.1a). These 7 TgACS candidates were further validated by bioinformatic analysis including sequence alignment and phylogeny (Fig.1b, Fig.2, Fig.S1). Multiple sequence alignment using Clustal Omega online tool (https://www.ebi.ac.uk/Tools/msa/clustalo) showed that the all of 7 putative TgACS candidates possess consensus amino acid sequences of canonical ACS motifs 1-IV and partly of motif V (Fig.1b, Sup 1). Phylogenetic analysis clustered the different TgACSs to specific clusters based on their homology to other eukaryotic counterparts (Fig.2). Of note, TgACS1 cladded together with the human ACSs belonging to the bubble-gum gene family (ACSBG), also known to be involved in β-oxidation[START_REF] Pei | The Acyl-CoA Synthetase 'Bubblegum' (Lipidosin)[END_REF]. TgACS2, TgACS3, TgACS4 and TgACS5 clustered in the long chain acyl-CoA synthetase (or acyl-CoA ligases) group within a coccidian/chromerida super-clade (Fig.2). TgACS6 clustered with the human ACSS family, more specific to short acyl chains like propionyl-CoA[START_REF] Watkins | Evidence for 26 Distinct Acyl-Coenzyme A Synthetase Genes in the Human Genome[END_REF] (Fig.2) Interestingly, TgACS7 clustered away from all known ACSs from humans, A. thaliana and P. falciparum. Further sequence and phylogenetic analyses showed no direct homology/conservation of TgACS7 with the sequences of known homologs from humans, A. thaliana, or P. falciparum. However, unique conserved homologs of TgACS7 were identified within the coccidians and the chromerids (close photosynthetic relatives of apicomplexans), Hammondia hammondi, Cryptosporidium parvum, Neospora_caninum, and Chromera velia using

  LACS1 alongside complementary assistance from LACS4 and LASC2. Male fertility is governed together by the function of LACS1 and LACS4 while as oil biosynthesis is controlled by combination of LACS1, LACS4 and LACS9[START_REF] Zhao | Functional Overlap of Long-Chain Acyl-CoA Synthetases in Arabidopsis[END_REF]. (Cvel_9659), C. velia (Cvel_3410), C. velia (Cvel_2306), C. velia (Cvel_26948) ; Cryptosporidium parvum (cgd5_3200), C. parvum (cdg3_2870), C. parvum (cdg3_2870) C. parvum (cdg3_640) ; Hammondia hammondi (HHA_310150), H. hammondi (HHA_310080), H. hammondi (HHA_276155), H. hammondi (HHA_232580), H. hammondi (HHA_297220), H. hammondi (HHA_243800), H. hammondi (HHA_247760); Neospora caninum (NCLIV_063970), N. caninum (NCLIV_006300), N. caninum (NCLIV_006990), N. caninum (NCLIV_054250), N.

  an inducible knockdown parasite line for the gene TgACS2 and TgACS3 plasmid pPR2-DHFR (Katris et al. 2014) was used. For homologous recombination in RH-ΔKu80 TATi strain two separate homology flanks/regions were chosen. The 5' flank was amplified 1298 to PacI and NdeI digested vector pPR2 using NEB assembly reaction. Next, the 3' flank was amplified as a 1263 bp fragment beginning at the start codon of TgACS3 with the primers forward 5′-TGTTCCAGATTATGCCTTACCCGGGATGGCACTCCAGTACGCCTACC-3' and reverse 5′-TGGAGCTCCACCGCGGTGGCGGCCGCCTGCTTCGAGGAGAATGGCTTTC-3'. The 3' homology flank was annealed to XmaI and NotI digested pPR-HA3-DHFR vector that already contained the TgACS3 5' flank. The final cloned vector positions the start codon of TgACS3 downstream of the ATc-regulatable t7s4 promoter and a 3xHA tag. Similar protocol was used for construction of TgACS2-pPR2-DHFR plasmid. For the same, a 1535 bp 5with NotI and transfected this into TATiΔku80 parasites. Parasites were selected with the drug pyrimethamine and cloned out by limiting dilution. Screening of parasite clones TgACS3-iKD where the t7s4 promoter had successfully replaced the native TgACS3 promoter, was done using the primers P1 5'-CGATGACCTGTGTCGACCTGT P2 5′-TCTTCTTTGAGGGAAGAGGAAACG, P3 5'-GGTACCGAGCTCGACTTTCAC, P4 5'-CAGCTGATCGGAGGTTGGTCT and P5 5'-CTCCACCGTTTCCGGTTCCGT in the combinations described in supplementary fig S2. All PCRs were performed with TaKara Primestar Max polymerase. The knockdown of TgACS3 was induced with 0.5 μg ml -1 of anhydrotetracycline (ATc).

FIGURESFig. 1 .

 1 FIGURES

Fig. 3 .

 3 Fig.3. Endogenous localizations of the Toxoplasma ACSs a) The 7 TgACSs were localized endogenously at their C-terminus with 3×HA. Immunofluorescence with anti-HA antibody shows that all seven proteins localize to non-overlapping compartments within the parasite: TgACS1 as cytoplasmic vesicular ; TgACS2 at the vicinity of mitochondria ; TgACS3 within the parasite cytoplasm, perinuclear ; TgACS4 as reminiscent of endoplasmic reticulum (ER) ; TgACS5 at the basal end of the parasite ; TgACS6 dispersed throughout parasite cytoplasm and TgACs7 showing vesicular localization within cytoplasm. Towards the right side of the IFA images is depicted the phenotype scores of all 7 TgACSs based on CRISPR-cas9 mediated whole genome screen in T. gondii(Sidik et al. 2016). b) Western blot analysis using anti-HA antibody shows all TgACSs migrating at their predicted protein sizes on ToxoDB (except TgACS2).

Fig. 4 .

 4 Fig.4. TgACS2 localizes to mitochondrial vicinity and is dispensable for growth of T. gondii.a) Images generated using confocal microscopy depict that TgACS2 (HA-red) and mitochondrial marker (TOM40green) co-localize with each other, thereby confirming mitochondrial vicinity of this ACS. b) 3D reconstruction of confocal images was obtained for TgACS2/mitochondrial co-location, using UCSF ChimeraX software. c) Western blot analysis using anti-HA antibody showing a size difference of approx. 10 kDa between TgACS2 tagged at Cterminus (endogenous promoter) and at the N-terminus (Tet-based promoter). d) Western blot and IFA (e) using anti-

Fig. 5 .

 5 Fig.5. TgACS3 is required for parasite growth in vitroWestern blot (a) and IFA (b) analysis using anti-HA antibody show complete loss of TgACS3, 48 h after induction with ATc. c) Growth assays show reduction in plaque area upon TgACS3 depletion (+ATc), which is aggravated more in the presence of increased FBS content (10%), depicting effect of lipotoxicity in TgACS3-ikD (+ATc). d) The plaque area percentage of TgACS3-iKD (+ATc) declines by 4-5% in 0 and 1% FBS; by 8% in 10% FBS. e) The TgACS3-iKD showed a replication defect in the presence of ATc, with a significant decrease in the vacuoles with 7-10 parasites and consequent increase in vacuoles with 3-6 parasites. f) TgACS3-iKD + ATc parasites also exhibited a minor yet significant egress defect upon induction with calcium ionophore A23187. Three biological replicates were used per experiment (n=3). The error bars are representative of standard error of mean (SEM) for each study. Statistical significance was determined for each experiment by t-test using GraphPad Prism. Range of statistical significance was signified as per the p value, wherein 0.01-0.05=*, 0.01-0.001=** and <0.001=***.

Fig. 6 .

 6 Fig.6. TgACS3 depletion results in specific decrease in the phospholipid fatty acid species within the parasite. a) The total fatty acid (FA) content depicted by nmol FA/number of parasites, of TgACS3-iKD parasites showed no major difference in the presence of +ATc (red) or absence of -ATc (grey). b) The overall FA composition of TgACS3-iKD parasites depicted by mol%, show a minor yet significant increase in oleic acid (C18:1). TgACS3 depletion results in a significant decrease in the relative abundance (+ATc/-ATc) of total phospholipids (c) and possible consequent increase of free fatty acids (d). e) Graph showing slight increase in the relative abundance of triacylglycerols (TAGs).

Fig. S2 .

 S2 Fig. S2. Schematic of molecular strategy for generation of TgACS3-iKD tagged with 3*HA at its N-terminus. b) Confirmation of correct promoter replacement in the TgACS3-iKD line. Primers combinations during the screening PCR yielded products of expected amplicon size validating the mutant. The template genomic DNA was extracted from TgACS3-iKD line (M) and RH∆ Ku80 TATi strain (WT) (control). Primers 5+4 generated 1452 bp amplicon with RH∆ Ku80 TATi strain (WT) and approx. 4500 bp using TgAC3-ikD gDNA (M), suggesting incorporation of tetracycline inducible elements along with DHFR cassette at the genome locus of TgACS3. Expectedly, primers combinations specific to Tet-regulatable elements (1+2, 3+4) generated an amplicon approx. 3000 bp in size only using TgACS3 (M) as the gDNA template.

d

  Knockout of apicoplast TgATS2 disrupts LPA/PA for DrpC recruitment during cytokinesis d T. gondii can sense host environment and adapt to low host nutritional content d Under lipid starvation, parasite FASII and other lipid metabolic genes become essential d Upon nutrient deprivation, T. gondii induces host organelle remodeling and vesiculation Authors Souad Amiar, Nicholas J. Katris, Laurence Berry, ..., Geoffrey I. McFadden, Yoshiki Yamaryo-Botte ´, Cyrille Y.

Figure 1 .

 1 Figure 1. TgATS2 Is an Apicoplast Lysophosphatidic Acid Acyltransferase Important for Parasite Proliferation (A and B) IFA of stable TgATS2-HA expressing parasites (A) and transient TgAGPAT-HA expression (B). CPN60, apicoplast marker; TOM40, mitochondrial marker. Scale bars, 2 mm. (C) Expression of TgATS2 and TgAGPAT in LPAAT-deficient E. coli strain SM2-1. SM2-1DplsC E. coli mutant transformed with TgATS2 (1, 2), TgAGPAT (1, 2), TgAGPAT DNter1-72 , EcplsC, or empty pQE30Xa expression vector were grown at 30 C (permissive) or 42 C (non-permissive) for 20 h (n = 3). (D and E) Confirmation of TgATS2-HA loss by western blot analysis in TgATS2-KI (D) and TgATS2-KO (E) using anti-HA (anti-Gra1, loading control). (F) Confirmation of TgATS2-HA signal loss in DTgATS2 by IFA using anti-HA. Scale bars, 2 mm. (G) Plaque assay showing a mild growth defect in DTgATS2 mutants. (H) Cell-based growth fitness assay confirmed the growth defect in the DTgATS2 mutants 30 h post-infection (n = 3). (I) Proliferation assay confirmed a replication defect in DTgATS2 mutants (n = 3). *p % 0.05, **p % 0.01, ***p % 0.001, and ****p % 0.0001.

Figure 2 .

 2 Figure 2. Disruption of TgATS2 Induces Parasite Cytokinetic Defect, Residual Body Enlargement, and Organelle Segregation Deficiency

  (A) IFA of DTgATS2-HA and parental line using anti-HA and anti-IMC1 shows that DTgATS2-HA has a cytokinetic defect phenotype (scale bars, 2 mm). (B) IFA of DTgATS2 and parental line transiently expressing MORN1-mCherry (IMC basal tip) and anti-IMC1 (scale bars, 2 mm). (C and D) Confirmation of enlarged residual bodies in DTgATS2 by IFA using anti-GAP45 (IMC marker) (C) and by statistical analysis of residual body size (D). Scale bars, 2 mm. (E) IFA observation of extracellular parasites using anti-SAG1 reveals egressed parasites tethered at their basal ends (white arrowhead, PM tether). Scale bars, 2 mm.

Figure 3 .

 3 Figure 3. Lipidomic Analysis of DTgATS2 Mutant (A) Fatty acid composition of total lipid extracted 72 h post-infection. (B) is enlargement of A. (C) Relative fatty acid abundance of DTgATS2 to the parental line. (D) Relative major phospholipid abundance of DTgATS2 to parental line. (E) LPA/PA ratio. (F-H) Individual molecular species of LPA (F), PA (G), and DAG (H). Fatty acids are shown as Cx:y, where x is the number of carbons and y is the number of unsaturations. n = 4; *p % 0.05, **p % 0.01, ***p % 0.001, and ****p % 0.0001.

Figure 4 .

 4 Figure 4. DTgATS2 Induces the Specific Mis-localization of TgDrpC, a Dynamin-Related Protein Involved in Endodyogeny, Leading to Cytokinetic Defects during Tachyzoite Division (A) IFA localization of TgDrpC-HA expressed in parental line shows ring structures at the growing ends of daughter cells during division (top panel) but fails to do so when expressed in DTgATS2 (bottom panel). Scale bars, 2 mm. (B) TgDrpC-HA localization during tachyzoite division cycle in the parental line (top panel) and its mis-localization in DTgATS2 mutant. Scale bars, 2 mm. (C) IFA localization of TgESP15, a known interactor of TgDrpC, in DTgATS2 and parental line using anti-HA and anti-IMC1. Scale bars, 2 mm. (D) IFA localization of TgDrpC and TgDrpC-DPA domain-Cas9-RFP using anti-HA and anti-IMC1 (scale bars, 2 mm) reveals the mis-localization of TgDrpCDPA domain during endodyogeny.

Figure 5 .

 5 Figure 5. Changes in Host Nutritional Environment Induces an Upregulation of the Apicoplast FASII Metabolic Capacities in T. gondii Tachyzoites and P. falciparum Blood Stages and Are Pivotal for Enzymes Involved in Metabolic Adaptation

Figure 6 .

 6 Figure 6. Nutrient Starvation Unveils the Formation of Multi-vesicular Bodies from Host Cell Organelles, Whose Content Is Imported toward Parasites

Figure 7 . 30 ,

 730 Figure 7. Proposed Model for Cytokinesis, Lipid Acquisition, and Metabolic Adaptation under Adverse Host Lipid Environment in T. gondiiLeft: under lipid-rich environment, T. gondii can readily acquire FAs and lipids by de novo synthesis (apicoplast) and host cell scavenging. The apicoplast ATS2 generates PA and regulates the balance of LPA/PA, necessary for DrpC. Right: in a host lipid-starved environment, the parasite adapts its metabolism by increasing FASII to produce more fatty acids to compensate their absence from the host cell. Concomitantly, the parasite induces morphological changes in the host to increase scavenged resources, including the nucleus, ER, and gMVBs.

  . Mutant clones with successful replacement of the native TgACS gene locus with the resistant cassette and inducible promoter together with CDS was confirmed by PCR using their genomic DNA as template and following primers as

FIGURE 1 Fig. 1

 11 FIGURE 1

FIGURE 2 Fig. 2 Generating

 22 FIGURE 2

FIGURE 3 Fig. 3

 33 FIGURE 3

FIGURE 4 Fig. 4

 44 FIGURE 4

FIGURE 5 Fig. 5

 55 FIGURE 5

FIGURE 6 Fig. 6 FIGURE 7 Fig. 7

 6677 FIGURE 6

Figure 1 .

 1 Figure 1. Determination of the best P. falciparum. α-tubulin conformations for in-silico screening. (A) Conformations of the H1-S2 loop (residues 35-60) of P. falciparum α-tubulin, showing the overlap of the initial homology model based on homology predictions to mammalian tubulin (cyan) and lowestenergy S4MPLE (Sampler for Multiple Protein-Ligand Entities)-generated open-site geometry model, which takes into account the dinitroaniline binding ability (orange). The residues Ser6, Ile235 and Leu167 in the dinitroaniline binding site of the P. falciparum tubulin that were found involved in interactions for every conformation and used as criteria for selection are coloured in red, and their immediate neighbourhood (within 4Å) is in green. The loop in the homology model-in cyan-is seen to block access to the site. When displaced to allow dinitroaniline binding ability the predicted new position (in orange) opens access to the three key residues. (B) View of the "red" key residues in a protein surface model, showing that they (and their immediate neighborhood, in green) are buried in the homology model (upper panel) but accessible in the sampled conformer model (lower panel).

Figure 2 .

 2 Figure 2. Effect of selected compounds on plant cell microtubule network organization. BY2 cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. CM539 is an example of a compound that was found inactive. Bar = 20 µm.

Figure 1 .

 1 Figure 1. Determination of the best P. falciparum. α-tubulin conformations for in-silico screening. (A) Conformations of the H1-S2 loop (residues 35-60) of P. falciparum α-tubulin, showing the overlap of the initial homology model based on homology predictions to mammalian tubulin (cyan) and lowest-energy S4MPLE (Sampler for Multiple Protein-Ligand Entities)-generated open-site geometry model, which takes into account the dinitroaniline binding ability (orange). The residues Ser6, Ile235 and Leu167 in the dinitroaniline binding site of the P. falciparum tubulin that were found involved in interactions for every conformation and used as criteria for selection are coloured in red, and their immediate neighbourhood (within 4Å) is in green. The loop in the homology model-in cyan-is seen to block access to the site. When displaced to allow dinitroaniline binding ability the predicted new position (in orange) opens access to the three key residues. (B) View of the "red" key residues in a protein surface model, showing that they (and their immediate neighborhood, in green) are buried in the homology model (upper panel) but accessible in the sampled conformer model (lower panel).

17 Figure 1 .

 171 Figure 1. Determination of the best P. falciparum. α-tubulin conformations for in-silico screening. (A) Conformations of the H1-S2 loop (residues 35-60) of P. falciparum α-tubulin, showing the overlap of the initial homology model based on homology predictions to mammalian tubulin (cyan) and lowestenergy S4MPLE (Sampler for Multiple Protein-Ligand Entities)-generated open-site geometry model, which takes into account the dinitroaniline binding ability (orange). The residues Ser6, Ile235 and Leu167 in the dinitroaniline binding site of the P. falciparum tubulin that were found involved in interactions for every conformation and used as criteria for selection are coloured in red, and their immediate neighbourhood (within 4Å) is in green. The loop in the homology model-in cyan-is seen to block access to the site. When displaced to allow dinitroaniline binding ability the predicted new position (in orange) opens access to the three key residues. (B) View of the "red" key residues in a protein surface model, showing that they (and their immediate neighborhood, in green) are buried in the homology model (upper panel) but accessible in the sampled conformer model (lower panel).

Figure 2 .

 2 Figure 2. Effect of selected compounds on plant cell microtubule network organization. BY2 cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. CM539 is an example of a compound that was found inactive. Bar = 20 µm.

Figure 2 .

 2 Figure 2. Effect of selected compounds on plant cell microtubule network organization. BY2 cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. CM539 is an example of a compound that was found inactive. Bar = 20 µm.

Figure 3 .

 3 Figure 3. Effect of selected compounds on mammalian cell microtubule network organization. HeLa cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. Bar = 20 µm.

Figure 3 .

 3 Figure 3. Effect of selected compounds on mammalian cell microtubule network organization. HeLa cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. Bar = 20 µm.

Figure 4 .

 4 Figure 4. Comparison of the effects of CM094, CM571 and CM852 on mammalian and plant microtubule polymerization in vitro. (A) Pure bovine brain tubulin polymerization assay. Tubulin was allowed to polymerize at 37 °C. Fluorescence of DAPI bound to microtubules was measured to monitor microtubule polymerization, as described in the Material and Methods section. Experiments were performed in triplicate, in the presence of the indicated compounds. Results are presented as mean ± standard error of the mean (SEM). Effect of dinitroaniline (B), CM571 (C), CM852 (D), and CM094 (E) on soybean tubulin assembly. Recombinant soybean tubulin was polymerized as described in the Material and Methods section, in the presence of DMSO (control) or the indicated compounds.

Figure 4 .

 4 Figure 4. Comparison of the effects of CM094, CM571 and CM852 on mammalian and plant microtubule polymerization in vitro. (A) Pure bovine brain tubulin polymerization assay. Tubulin was allowed to polymerize at 37 • C. Fluorescence of DAPI bound to microtubules was measured to monitor microtubule polymerization, as described in the Material and Methods section. Experiments were performed in triplicate, in the presence of the indicated compounds. Results are presented as mean ± standard error of the mean (SEM). Effect of dinitroaniline (B), CM571 (C), CM852 (D), and CM094 (E) on soybean tubulin assembly. Recombinant soybean tubulin was polymerized as described in the Material and Methods section, in the presence of DMSO (control) or the indicated compounds.

Figure 5 .

 5 Figure 5. Structure of the selected compounds.

Figure 5 .

 5 Figure 5. Structure of the selected compounds.

Figure 6 .

 6 Figure 6. Effect of selected compounds on T. gondii and P. falciparum. (A) The effect of the compounds on T. gondii replication within human fibroblasts was tested. The graph presents T. gondii growth inhibition curves for each treatment at concentrations ranging from 0.01 to 100 µM, in triplicates. The quantity of individual parasites within intracellular vacuoles exposed or not to the drug for 30 h postinvasion was automatically scored. The calculated IC50 is indicated for each compound. (B) Plaque assays showing at 4 and 7 days the expansion of HFF cleared zones due to successive rounds of lytic cycle that were initiated by a single parasite. HFF monolayers plated on a 6-well plate were inoculated with 200 invasive tachyzoites per well for 1 h. The non-invading tachyzoites were washed away and cells were incubated with medium containing the vehicle (DMSO, 0) or different concentrations of CM094. After fixation and Crystal violet staining, cells were air dried and scanned before image processing and plaque area measurement. At day 7, post-inoculation, a highly significant reduction of parasite expansion was observed for the cultures exposed to 15 µM of CM094 and no plaque was detected at 30 µM while the HFF monolayer was well preserved. (C) Histograms showing the cumulated area of HFF cleared zones. p value = 0.0082. (D)The effect of selected compounds on P. falciparum intra-erythrocytic growth was tested. The growth of parasites within human red blood cells is determined using a classical SYBR Green assay followed by quantification of parasite DNA fluorescence. The graph presents growth curves for concentrations of each compounds ranging from 0 to 250 µM, in triplicates.

Figure 6 .

 6 Figure 6. Effect of selected compounds on T. gondii and P. falciparum. (A) The effect of the compounds on T. gondii replication within human fibroblasts was tested. The graph presents T. gondii growth inhibition curves for each treatment at concentrations ranging from 0.01 to 100 µM, in triplicates. The quantity of individual parasites within intracellular vacuoles exposed or not to the drug for 30 h post-invasion was automatically scored. The calculated IC 50 is indicated for each compound. (B) Plaque assays showing at 4 and 7 days the expansion of HFF cleared zones due to successive rounds of lytic cycle that were initiated by a single parasite. HFF monolayers plated on a 6-well plate were inoculated with 200 invasive tachyzoites per well for 1 h. The non-invading tachyzoites were washed away and cells were incubated with medium containing the vehicle (DMSO, 0) or different concentrations of CM094. After fixation and Crystal violet staining, cells were air dried and scanned before image processing and plaque area measurement. At day 7, post-inoculation, a highly significant reduction of parasite expansion was observed for the cultures exposed to 15 µM of CM094 and no plaque was detected at 30 µM while the HFF monolayer was well preserved. (C) Histograms showing the cumulated area of HFF cleared zones. p value = 0.0082. (D)The effect of selected compounds on P. falciparum intra-erythrocytic growth was tested. The growth of parasites within human red blood cells is determined using a classical SYBR Green assay followed by quantification of parasite DNA fluorescence. The graph presents growth curves for concentrations of each compounds ranging from 0 to 250 µM, in triplicates.

4. 4 .

 4 In Silico Screening on Alpha-Tubulin 3D-Model: Virtual Library of Compounds, Molecular Docking and Processing Methods

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'Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation', Elmendorf, H. G. and Haldar, K. (1994) 'Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes', Journal of Cell Biology. doi: 10.1083/jcb.124.4.449. Fang, J., Marchesini, N. and Moreno, S. N. J. (2006) 'A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol', Biochemical Journal, 394(2), pp. 417-425. doi: 10.1042/BJ20051393. Ferguson, D. J. P. et al. (1974) 'Ultrastructural Study of Early Stages of Asexual Multiplication and Microgametogony of Toxoplasma Gondii in the Small Intestine of the Cat', Acta Pathologica Microbiologica Scandinavica Section B Microbiology and Immunology, 82 B(2), pp. 167-181. doi: 10.1111/j.1699-0463.1974.tb02309.x. Fleige, T. et al. (2007) 'Carbohydrate metabolism in the Toxoplasma gondii apicoplast: Localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator',

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  Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling Souad Amiar,1,5 Nicholas J. Katris,1,5 Laurence Berry, 2 Sheena Dass, 1 Samuel Duley,1 Christophe-Sebastien Arnold, 1 Melanie J. Shears,3 Camille Brunet, 1 Bastien Touquet,4 Geoffrey I. McFadden, 3 Yoshiki Yamaryo-Botte ´,1,6, * and Cyrille Y. Botte ´1,6,7, * 1 ApicoLipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Universite ´Grenoble Alpes, INSERM U1209, Grenoble, France 2 Dynamique des interactions Membranaires normales et pathologiques, UMR5235, Universite ´Montpellier II, Montpellier, France 3 McFadden Laboratory, School of Biosciences, University of Melbourne, Melbourne, VIC 3010, Australia 4 Team Cell and Membrane Dynamics of Parasite-Host Interaction, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR5309,

		Botte Ćorrespondence
	Universite ´Grenoble Alpes, Grenoble, France
	5 These authors contributed equally 6 Senior author	yoshiki.yamaryo@gmail.com (Y.Y.-B.),
	7 Lead Contact	cyrille.botte@univ-grenoble-alpes.fr
		(C.Y.B.)
		In Brief
		Apicoplast de novo lipid synthesis and
		lipid host scavenging are both critical for
		apicomplexan intracellular development.
		Amiar et al. show that the parasite adapts
		to the fluctuations of host nutritional
		content to regulate the metabolic activity
		of both apicoplast and scavenging
		pathways and maintain parasite
		development and division.

*Correspondence: yoshiki.yamaryo@gmail.com (Y.Y.-B.), cyrille.botte@univ-grenoble-alpes.fr (C.Y.B.) https://doi.org/10.1016/j.celrep.2020.02.072

error bars are representative of standard error of mean (SEM) for each study. Statistical significance was determined for each experiment by t-test using GraphPad Prism. Range of statistical significance was signified as per the p value, wherein 0.01-0.05=*, 0.01-0.001=** and <0.001=***.

Cell Reports 30, 3778-3792, March 17, 2020 ª 2020 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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C incoporation in the M+12 and M+14 isotopologues in the TgACS knock-down mutant. by guest, on April 22, 2018 www.jlr.org Downloaded from

C-glucose (800μM final concentration) was added to the cultures for

C-U-acetate labelling experiment. All lipids were then analysed by GC-MS after derivatisation using Methprep II (Alltech). Mass shift in each mass spectra of each fatty acids were analysed to assess the incorporation of 13 C to fatty acids. by guest, on April 22, 2018 www.jlr.org Downloaded from

ACKNOWLEDGMENTS

We would like to thank Prof. Alan Cowman and Dr. Ali Hakimi for sharing parasite strains, reagents, and fruitful advice. This work and C.Y.B., Y.Y.-B., S.A., N.J.K., and C.B. are supported by Agence Nationale de la Recherche, France (grant ANR-12-PDOC-0028, Project Apicolipid), the Atip-Avenir and Finovi programs (CNRS-INSERM-FinoviAtip-AvenirApicolipid projects), and Laboratoire d'Excellence Parafrap, France (grant ANR-11-LABX-0024). C.Y.B. and G.I.M. are supported by the LIA-IRP CNRS Program (Apicolipid project).

Acknowledgments

This work was supported by Agence Nationale de la Recherche, France (Grant ANR-12-PDOC-0028-Project Apicolipid), the Atip-Avenir and Finovi programs (CNRS-INSERM-Finovi Atip-Avenir Apicolipid projects), and the Laboratoire d'Excellence Parafrap, France (grant number ANR-11-LABX-0024). CYB is a CNRS Atip-Avenir Fellow.

Acknowledgments:

The authors thank Magali Audry for her input in the initial steps of modelisation, Didier Rognan and Esther Kellenberger for having trained Loraine Brillet-Guéguen in molecular docking, Gilles Bisson for the scoring function which allowed to select the 5 tubulin conformations for docking, Aline Thomas for help on several molecular modeling softwares, Olivier Bastien for introduction on R scripting, the ChemAxon company (http://www.chemaxon.com) for having allowed academics to freely use their software, especially the Standardizer (version of 2006) for structure canonicalization and transformation of the ligands before the docking steps, Boris Striepen (Univ. Pensylvania, PA, USA) for providing the T. gondii RH strain expressing GFP, Karin Sadoul for critical reading of the manuscript. Part of this work has been performed at the CMBA molecular screening platform (Grenoble, France) which is a member of the GIS-IBiSA and the infrastructure ChemBioFrance.

Protein sequence analysis and Phylogeny: Identification of TgACSs

Members of the Acyl CoA synthetase (ACS) gene family within T. gondii were identified using various online bioinformatic tools, Clustal omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), (https://www.uniprot.org), EuPathDB (https://eupathdb.org/eupathdb). A phylogenetic tree of ACS proteins in several eukaryotes including apicomplexa, humans and plants was created using the online platform Phylogeny.fr. The organisms used for ACS protein sequences for generation of the phylogenetic tree include: T. gondii_TgACS1 (TGGT1_297220), T. gondii_TgACS2 (TGGT1_310150), T. gondii_TgACS3 (TGGT1_310080), T. gondii_TgACS4 (TGGT1_243800), T. gondii_TgACS5 (TGGT1_247760), T. gondii_TgACS6 (TGGT1_232580), T. gondii_TgACS7 (TGGT1_276155), P. falciparum_PfACS1a (PF3D7_1479000), P. falciparum_PfACS2 (PF3D7_0301000), P. falciparum_PfACS3 (PF3D7_1253400), P. falciparum_PfACS4 (PF3D7_1372400), P. falciparum_PfACS5 (PF3D7_0731600), P. falciparum_PfACS6 (PF3D7_0401900), P. falciparum_PfACS7 (PF3D7_1200700), P. falciparum_PfACS8 (PF3D7_0215300), P. falciparum_PfACS9 (PF3D7_0215000), P. falciparum_PfACS10 (PF3D7_0525100), P. falciparum_PfACS11 (PF3D7_1238800), P. falciparum_PfACS12 T. gondii ACBP1 iKD This study N/A T. gondii ACBP2 iKD This study N/A T. gondii ACBP1/ACBP3 double KO This study N/A T. gondii ACBP1/SCP2 double KO This study N/A T. gondii ATS2-HA This study N/A T. gondii ATS2-KI This study N/A T. gondii ATS2-KO This study N/A

DATA AND CODE AVAILABILITY

No unique code or software was generated in this study. All datasets generated and analyzed during this study are available upon request to the lead contact Cyrille Botte (cyrille.botte@univ-grenoble-alpes.fr).

CHAPTER II: LIPID METABOLISM IN APICOMPLEXAN PARASITES

France. * Equal senior and corresponding authors. To whom correspondence should be sent, cyrille.botte@univgrenoble-alpes.fr / cyrille.botte@gmail.com; yoshiki.botte-yamaryo@univ-grenoble-alpes.fr ABSTRACT Apicomplexa comprise several pathogenic protists, that heavily depend on lipid metabolism for the survival within their hosts. The lipid synthesis within these parasites relies on an essential combination of fatty acids obtained from de novo synthesis and scavenging from the host. The metabolic utilization of these fatty acids is mediated by acyl-activating enzymes. These include acyl CoA synthases (ACS) that catalyze the activation of fatty acids, primarily through the formation of a thioester bond. The role of these enzymes in FA utilization in T. gondii has been unforeseen. Here, we identified 7 putative ACSs encoded by the genome of T. gondii (TgACS).

Interestingly, the TgACSs localize to different sub-cellular compartments of the parasite, suggesting their exclusive functions. The perinuclear/cytoplasmic TgACS3 regulates replication and growth of Toxoplasma tachyzoites. Lipidomic analysis of parasites lacking TgACS3, suggests its role in activation of FAs directed towards parasite phospholipid synthesis. Altogether, the results presented here describe a putative ACS family of enzymes in T. gondii potentially involved in FA activation and metabolism. In this study, I assessed the role of nutrient starvation on host cells and intracellular parasites, separately by analysing the lipid droplets and a host-specific mitochondrial lipid anti-lyso-bi-phosphatidic acid (LBPA). The import of lipid droplets was increased within the parasite was upregulated in conditions of FBS starvation. Host LBPA was found surrounding the PVM in the infected host cell, within the PV and the parasite, but its localization and intensity remained unchanged in response to reduced FBS content. (Mancio-Silva et al., 2017).

Supplementary figures

Together, our results reveal the central role of the apicoplast to provide specific precursors for membrane biogenesis during cytokinesis and, most important, to be a central metabolic hub to adapt the parasite metabolic capacities upon nutrient availability and environmental changes. The data also point to major modifications in vesiculation and the use and scavenging of these membrane structures by the parasite upon such environmental changes. The data also corroborate recent results showing that the mosquito lipid environment regulates the metabolic activity of transmissible sporozoites [START_REF] Costa | Non-competitive resource exploitation within mosquito shapes within-host malaria infectivity and virulence[END_REF]. The fundamental role of these physiological changes induced by the parasite in response to host environment provides novel insights into parasite biology and offers new avenues to explore in the fight against toxoplasmosis and malaria.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS

The authors declare no competing interests. Plasmodium falciparum and the chromerids, Chromera velia and Vitrella brassicaformis, and found putative candidates, PfACS (PF3D7_0627800, the enzyme likely responsible for the acetyl-CoA synthesis from acetate described in ( 22) CvACS (Cve1_1982) and VbACS (VBra_8944), respectively. We compared those to protein sequence of SeACS, TgACS, and human ACS, which showed that there is a high homology amongst these divergent organisms (Fig. S1). This, added to the fact that recombinant HsACS was shown to generate acetyl-CoA from acetate (35), strongly supports that the TgACS gene locus is encoding for an active ACS. We furthermore performed in silico protein threading (or homology modelling) against the crystal structure of S. enterica. Predicted TgACS structure can merge together with that of S. enterica and conserves the structural amino acids localization of predicted substrates binding Co-A and AMP with high scores and confidence from the prediction software. This high structural conservation is indicative of an important and fundamental requirement of ACS in the cellular function and homology to characterized ACS (Fig. 1B-F).

STAR+METHODS KEY RESOURCES

In order to localize TgACS, we generated a construct that expressed TgACS fused to a C-terminal triple haemaglutinin (3×HA) epitope-tag under control of an anhydrotetracycline (ATc)-regulated promoter (TgACS-HA-iKO, Fig. 2A,B,C). This TgACS-HA-iKO strain enables us to analyze the effect of disruption of TgACS. Two independent mutants were generated within a T. gondii TATi_∆Ku80 background (36), the successful replacement of the endogenous locus was confirmed via PCR (Fig. 2C). Immunofluorescence assays (IFA) using anti-HA antibody showed that TgACS-HA was localised within the cytosol of the parasite similarly to previous reports (30,37) (Fig. 3A, upper panels). The effect of repression of TgACS was observed by IFA after the addition of ATc (0.5 µg/mL) to the parasite culture (Fig. 3A, lower panels). The addition of ATc led to significant loss of HA signal within two days post after the treatment with ATc (Fig. 3A), suggesting that TgACS most likely is not essential for in vitro growth for parasites.

We then analysed the effect of ATc on the TgACS-HA protein level via western blot analysis. TgACS-HA protein was detected as a single distinct band with an apparent molecular mass of 80 kDa corresponding to its predicted molecular weight of 79.8 kDa. The addition of 0.5 µg/mL ATc to the culture medium down-regulated TgACS-HA expression and the protein was undetectable after three days of ATc treatment (Fig. 3B). This kinetics complements the rapid loss of TgACS-HA signal observed during IFA.

While the loss of TgACS-HA did not affect parasite morphology, we investigated possible growth defects through plaque assays. The knockdown of TgACS lead to no from the shift in isotopic mass dependent on the amount of 12 C carbons compared to the integration of 13 C carbon atoms. For example, myristic acid (C14:0) consists of a carbon backbone of 14 carbons in length, the most common isotope would contain only 12 C with a detectable mass-to-charge ratio of m/z=228, as no 13 C has been incorporated we call this isotope mass 0 (M0). Integration patterns vary from one carbon to fully labelled, incorporation of four 13 C leads to an increase in mass by four, M+4, m/z=232. These isotopic distributions allow us to determine the metabolic flux and processes involved in lipid biosynthesis.

To investigate the role TgACS in the de novo lipid synthesis, we first labelled TgACS-HA-iKO parasites with U-13 C-glucose for 4 days continuously with ATc to disrupt TgACS similarly to the condition analysed for the total fatty acid composition (Fig. 4). Labelling for 4 days of ATc treatment and labelling with U-13 C-glucose showed no significant alteration for 13 C incorporation for all the fatty acid species analysed (Fig. 5A). MID analysis of C14:0 showed the typical two by two mass increase up to the M+14 mass due to 13 C from 6 carbon glucose was metabolised to 2 carbon acetyl-CoA in the apicoplast (Fig. 5B). This two by two went up to M+14 to show the full synthesis of C14:0 by the apicoplast FASII as we previously reported (15). Similarly, the MID analysis of C20:0, showed the full synthesis up to M+14 and M+16, mainly, correlating the origin of C20:0 from the apicoplast FASII major products C14:0 and C16:0 (Fig. 5C) (7,15).

Since 4 days of labelling still includes 2 days of expression of active TgACS protein (Fig. 3B), most of the 13 C-Carbon integration may have occurred before the loss of TgACS potentially masking obvious difference between the knock-down mutant and its parental line.

To measure the direct effect of loss of TgACS, we grew parasites with or without ATc for 48 h prior to the addition of U-13 C-glucose (pre-treatment 2 days). Then parasites were incubated with U-13 C-glucose for the next 72 h. In this condition, overall incorporation of 13 C-Carbon integration was reduced to approximately half of 4-day labelling (Fig. 5D). Here again, there was no significant difference between ATc treated and non-treated parasites in the integration of 13 C-carbon in fatty acids from glucose. In addition, the integration pattern of 13 C to apicoplast generated fatty acids, C14:0, showed clear two by two increase of mass up to M+14 in both condition (Fig. 5E), suggesting there was no activation or alteration of FASII upon the loss of TgACS. MID analysis of C14:0, however, showed a significant reduction of CoA is toxic for the cell and needs to be detoxified. One alternative way to degrade it would be through its utilization as a substrate in place of acetyl-CoA, which would potentially explain the +1 increase that we measured (Fig. 6).

Interestingly, accumulation of acetate in rats has been shown to down-regulate the expression of lipogenic genes such as (a) pyruvate kinase (PyK), which catalyses the synthesis of pyruvate for the generation of acetyl-CoA for fatty acid synthesis by the FAS, (b) acetyl-CoA carboxylase (ACCase), which catalyses the synthesis of malonyl-CoA the other essential substrate for the FAS pathways (both type I and II), and (c) FAS (43). All of these enzymes are present in T. gondii and could putatively be regulated by an accumulation of acetate following TgACS disruption; pyruvate kinase is present in two essential isoforms in T. gondii, TgPyKI, likely in the cytosol and TgPyKII dually localized in the mitochondrion and the apicoplast (44,45), ACCase is also present in two copies one in the apicoplast to fuel the FASII and one likely in the cytosol potentially for FASI and/or elongases (46).

Furthermore, it has also been shown that ACCase can be down regulated when there is the accumulation of the FASII products (47). So it is possible that the accumulation of C14:0 that is not elongated anymore due to the lack of acetyl-CoA provided by TgACS could indeed inhibit the FASII pathway as we observed in this work. Similarly, the abrogation of 13 C incorporation from acetate in C18:1 in the 2 days pre-treatment (Fig. 6E) might be explained by a massive scavenging of C18:1 from the external environment to compensate for the loss of its synthesis via the elongation pathway. Altogether our observations conclude that TgACS loss perturbed the fatty acid metabolism the causing modification of lipid balance in the induced knockout due to its role in providing acetyl-CoA for FA elongation.

One of the important questions that remain unanswered is the origin of the acetate used by TgACS. In human blood, it is said to contain 50-200 µM of acetate. Here in vitro, supplemented FBS also contains certain amount acetate (48), but this is possibly irrelevant because thus far, no transporter for acetate has been identified in Apicomplexa. Furthermore 13 C-Glucose labelling in P. falciparum shows that a small part of the intracellular acetate pool originates from glucose but that most of it is of unknown origin (22). Although acetate can be found in the extracellular environment, its origin in Apicomplexa might not be from importing it but maybe as a downstream product of host or parasite metabolism.

In summary, we demonstrated that (a) TgACS is a non essential enzyme present as a uniform protein of the cytosol and likely the nucleus, (b) it does not participate in providing substrate for the de novo synthesis of FA by the apicoplast FASII, (c) that its disruption does not have