I dedicate this thesis to the two people who would have been so proud, my grandmother and father ~For you Ma and Daddy~ ACKNOWLEDGMENTS I will eternally be grateful to my supervisors, Cyrille and Yoshiki. You have supported me constantly in my professional and personal life. I cherish the relationship we have developed in the past 3 years. Specifically, to you Yoshiki, thank you for your patience when teaching me lipidomics, it really was the highlight of my training here. I also express my gratitude to my thesis committee members for taking the time to participate in my defence; Malcolm McConville, Lilach Sheiner, Mohamed-Ali Hakimi and Olivier Épaulard.

Merci to Marie-France, Catherine, Delphine, and Pierre; thank you for sharing your coffee breaks and teaching me about French culture, it made my mornings very special. Thank you to the newest members during my time at the lab, Célia and Mitch (Nyamekye), who were always game for my lab shenanigans. I am grateful to Nick and Sheena, the people who were the most instrumental in my initiation of Toxoplasma and Plasmodium molecular and cellular biology. To my 'chouchou' Sarah, my little sister of the lab, thank you for listening to my stories and teaching me the difference between Haute-Savoie and Savoie (and that Reblochon is the best French invention ever).

Merci cher Samuel, tu as certainement été mon plus grand soutien dans l'apprentissage de la langue et de la culture françaises, tu as été un ami cher en le faisant. To the person in the lab that became my closest friend and confidant, Christophe (my PhD brother), thank you for lending me your ear (even when you were busy with experiments) and reassuring me that I wasn't going insane, it was just another symptom of doing a PhD. Aux Terraformeurs/Loulous ; Pignata, Poupou, Benjamin et Perrine ; merci pour tous nos repas, rires et bons moments. Vous m'avez accepté les bras ouverts dans vos vies et avez rendu ces 3 dernières années spéciales avec tous nos voyages et réunions. Je suis tellement reconnaissante de partager cette journée avec vous. To Yoan, thank you for sharing your own PhD/life experiences and making me feel that my emotions were valid. William, thank you for your advice on the introduction and for being my first real friend in France. To my best friend, Karasha even though we are 10 000 km apart, day or night you were only a phone call away when I needed a comforting voice or just a 'b%&ching' session.

A ma famille française ; Brigitte, Bernard, Yaya, Bilitis et Dimitri, merci de m'avoir accepté ainsi que ma famille tels que nous sommes. Vous avez fait de vos maisons un refuge loin du travail et vous m'avez fait me sentir comme une Brocco. Merci d'être patients avec mon français et de me nourrir jusqu'à ce que j'explose. À ma belle-mère, merci pour toutes nos discussions, de te préoccuper de mes ennuis et de mes émotions. A mes petits Brofillets préférés (même si vous ne le comprenez pas encore), Léon et Alice, merci de m'avoir fait oublier que je suis une adulte ; jouer avec vous est le meilleur remède anti-stress.

To my family back home, there are not enough words to describe my gratitude for your constant love and support whilst being so far away (Thank god for video calling!): Thatha, Indresan Mamma, Aunty Viloshnee, Caitlyn, Jordan, Saneshan Mamma, Aunty Poovesh, Daegan and Tanay. To my little brother Kreyan, thank you for your big brother advice on career and life choices. To the person that made me who I am; Mummy, I could never explain to you how much your relentless adoration and care has sustained me through the past 3 years (and of course way before and after this), and I thank you from the bottom of my heart.

Finally, and not least (since he absolutely deserves his own paragraph), Benjitto Brocco; my life-time adventure companion. If it were not for you, I most probably wouldn't be here writing this thesis. Thank you for letting me dump all my anxiety and drama on you every single day for the past 6 years. Thank you for understanding my limitations but still making me feel like a superhero. Thank you for making me laugh until I can't breathe, supporting my career goals and levelling with me when I needed it most. You are my everything and I can't wait for the forever. (PS: thank you for proofreading and translating!) You have all made my life in and out of the lab extremely special, and I am sure I will never forget my PhD years.

Apicomplexa comprise of unicellular pathogens that cause major diseases such as toxoplasmosis and malaria. These obligate intracellular parasites heavily depend on lipid metabolism for their survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs), obtained from de novo synthesis and host scavenging. Current data, points at the parasites being able to channel a constant, and otherwise toxic, flux of host FA to lipid storage; only to be used when needed, during parasite division stages. This suggests that the biogenesis of parasite lipid storage, i.e., lipid droplets (LD), and their lipid/FA content mobilization is a crucial process for the parasite. Little is known about the generation/utilization of the parasite LDs. The processing and assembly of FA into major LD lipid class, triacylglycerol (TAG), are key processes for parasite survival.

My PhD work focused on answering questions surrounding the lipid metabolism of Toxoplasma gondii tachyzoite and Plasmodium falciparum blood stage parasites. I did this by focussing on three protein families namely, acyl-CoA synthetases (ACSs), phospholipases and acyltransferases (ATs). Using lipidomics and metabolite flux analysis, we show that these proteins have key roles in the mobilization or biogenesis of LDs. This work reveals that their metabolic activity depends on host nutrient availability.

I confirm the function of an ACS in activating FA for metabolic utilization by the parasite. Specifically, TgACS1 functions as a TAG mobilizer and provides FAs to the parasite under low host nutrient environment, and more particularly during extracellular conditions. This protein is unique in that it contains a bubble-gum domain imperative for FA β-oxidation. Complementation studies now show insight into this previously understudied process in Toxoplasma parasites.

I then investigated another ACS, TgACS3, an essential protein that when absent leads to parasites death. This protein becomes even more crucial at a high host nutrient environment. Fluxomic analyses reveal that this protein has a preference in the metabolism of exogenous FFA that are usually redirected to TAG (LD) and PL synthesis. These 'back-to-back' papers illustrated the diverse functionality of the ACS protein family in T. gondii parasites and how their nutrient-dependent roles are central to TAG/LD metabolism.

Continuing with the theme of the nutrient adaptability, I studied a patatin-like phospholipase in schizont stage P. falciparum parasites. By lipidomic analyses I show this mitochondrial protein is responsible for the hydrolyses of phosphatidylglycerol to a lysobisphosphatidic acid. This balance seems to be important for schizont to ring conversion under lipid starvation. This work exemplifies that the lyso-phospholipid and phospholipid balance is crucial for the advancement of asexual parasites, but by changing their metabolic programme parasites can evade death but only in a nutrient rich environment.

The third and final study of my PhD, investigated the function of two acyltransferases; glycerol-3-phosphate acyltransferase (TgGPAT) and diacylglycerol acyltransferase 2 (TgDGAT2) that both localize to the endomembrane system and cytoplasm of the Toxoplasma parasite. TgGPAT and TgDGAT2 are essential in different nutrient conditions, but both play a pivotal role in neutral lipid synthesis, specifically LD biogenesis (TAG formation). The loss of these proteins resulted in an accumulation of free FAs that cannot be 'detoxified' by incorporating them into lipid droplets, leading to parasite death.

My thesis work provides novel insights to better understand the parasites' metabolic reprogramming tactics, which in turn give us clues on developing therapeutic strategies in combating the diseases they cause.

Le phylum des parasites apicomplexes comprend des pathogènes unicellulaires qui sont à l'origine de maladies telles que la toxoplasmose et le paludisme. La survie de ces parasites obligatoires unicellulaires est intimement liée à leur métabolisme lipidique. Leur anabolisme requiert une combinaison essentielle d'acides gras qui sont obtenu par néosynthèse ou détourné des ressources de son hôte. Les recherches actuelles indiquent que les parasites sont capables de canaliser les flux d'acides gras en provenance de l'hôte vers un mécanisme de stockage pour les utiliser lors du cycle reproductif et pour éviter les effets toxiques d'une accumulation. Cette observation suggère que le stockage de lipides sous forme de gouttelettes lipidique, et leur mobilisation sous formes d'acides gras sont cruciales au métabolisme du parasite. Les processus de synthèses des classes majeures de lipides et triglycérols à partir des acides gras sont essentiels à la survie des apicomplexes.

Mon travail de thèse s'est concentré sur le métabolisme lipidique des parasites Toxoplasma et Plasmodium falciparum au stade tachyzoite de la phase sanguine de leur cycle de vie. J'ai travaillé sur trois familles de protéines ; les acyl-CoA synthétases (ACSs), les phospholipases et les acyltransférases (ATs). En utilisant des méthodes lipidomique et d'analyse de flux métaboliques, nous montrons que ces protéines jouent un rôle clé dans la mobilisation ou la biogenèse des gouttelettes lipidiques. Ce travail indique que l'activité métabolique des parasites dépend de la disponibilité des nutriments pour l'hôte. J'ai confirmé la fonction d'une ACS dans l'activation des acides gras pour permettre leur intégration aux voies métaboliques du parasite. La protéine TgACS1 opère comme un mobilisateur de triglycérols pour fournir une source d'acides gras au parasite en conditions de stress nutritif, comme celles rencontrées lors de la phase extracellulaire du parasitaire. Cette protéine est unique puisqu'elle comporte également un domaine « bubble-gum » qui est essentiel pour catalyser les réactions de β-oxydation des acides gras. Nos expériences de complémentation démontrent l'existence de ce mécanisme jusqu'alors méconnu chez les parasites Toxoplasma.

J'ai ensuite étudié une autre ACS, TgACS3 qui est essentielle à la survie du parasite.

La fonction de cette protéine est cruciale dans un environnement riche en nutriments pour l'hôte. L'analyse des flux métaboliques a révélé que cette protéine utilise préférentiellement les acides gras libres d'origine exogène, qui sont habituellement redirigés vers la synthèse de triglycérols pour former des gouttelettes lipidiques et la synthèse de phospholipides. Nos deux publications successives illustrent les fonctions diverses des ACS chez le parasite T. gondii et la dépendance nutritive de leurs fonctions dans le métabolisme des triglycérols/gouttelettes lipidiques En continuant sur le thème de l'adaptabilité nutritive, j'ai étudié une phospholipase « patatin-like » présente lors du stade schizont chez les parasite P. falciparum. En utilisant des approches lipidomiques, nous avons démontré que cette protéine mitochondriale est responsable de l'hydrolyse du phosphatidylglycérol en acide lysobisphosphatidique. Cet équilibre semble important pour le passage du stade schizont au stade annulaire dans des conditions de carence lipidique. Ce travail illustre l'importance de la balance lyso-phospholipide/phospholipide pour la progression du développement asexuel du parasite ainsi que sa capacité à moduler son métabolisme pour survivre, mais seulement dans un environnement riche en nutriments.

La troisième et dernière étude conduite lors de ma thèse explore les fonctions de deux protéines : la glycérol-3-phosphate acyltransférases (TgGPAT) et la diacylglycérol acyltransférases 2 (TgDGAT2), qui sont toutes deux localisées dans le système endomembranaire et le cytoplasme des parasites Toxoplasma. TgGPAT et TgDGAT2 sont essentiels, dans des conditions nutritives différentes, mais jouent toutes deux un rôle primordial dans la synthèse des lipides neutre et notamment pour la synthèse de triglycérols. La déplétion de ces protéines entraine une accumulation d'acides gras libres non-activés qui ne peuvent être intégrés aux gouttelettes lipidiques et deviennent toxiques, entrainant la mort des parasites. A C-terminally tagged HA line with inducible post-translational protein degradation was generated using the Auxin-inducible Degron system (AID). Homology regions of the C-terminal end of TgGPAT/TgDGAT2 (not shown here) excluding the stop codon were amplified using the primers found in Table 1. PCR (Table 1) were preformed using mutant parasite DNA to show correct integration of the mAID sequence, the HX drug cassette and correct gene insertion. .

Table of Introductory Figures:

An introduction to the phylum of Apicomplexa

The 'super-phylum' alveolates is host to three eukaryotic phyla; Dinoflagellata, Ciliophora (Ciliata), and Apicomplexa (Sporozoa) [START_REF] Adl | The revised classification of eukaryotes[END_REF]. The feature unifying these organisms is the presence of a unique membrane structure forming flattened vesicles, called 'cortical alveoli', tightly apposed and maintained underneath the plasma membrane through a network of underlying sub-pellicular microtubules (Gould, Waller and McFadden, 2008). This structure allows for the organism's motility and is termed in inner membrane complex (IMC) in apicomplexa parasites.

Apicomplexa (with over 6000 species) are unlike their related alveolates since they have embraced an obligate intracellular parasitic lifestyle. Apicomplexa are comprised of five dominant groups of morphologically and ecologically diverse protists including several pathogenic protists [START_REF] Leander | Handbook of the Protists[END_REF]; a) Gregarinida, which inhabit invertebrate marine freshwater and terrestrial intestines, coelems and reproductive vesicles. They are potentially threatening to insect farms and laboratory colonies [START_REF] Leander | Handbook of the Protists[END_REF].

b)

Piroplasmida, which are named after their pear-shaped blood stages. They include organisms which are responsible for cattle-affecting diseases such as babesiosis (Babesia bovis) and theileriosis (Theileria parva/annulata). These parasites require a tick (Ixodidae) vector to facilitate transmission of the diseases [START_REF] Jalovecka | The Complexity of Piroplasms Life Cycles[END_REF].

c)

Cryptosporidida has come into much-needed limelight in recent years as the second most common cause of infant diarrhoea 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]. These parasites have a unique epicellular parasitic lifestyle, developing their parasitophorous vacuole (i.e. the intracellular niche within which apicomplexan develop in the host cell) just on the surface of the invaded host cell.

d)

Haemosporidia is comprised of three genera: Haemoproteus, Leucocytozoon, and Plasmodium. The later genus is responsible for malaria infection that can be considered one of the oldest pandemics.

e)

Coccidia, this is the largest group of apicomplexa protozoa which houses parasites such as Neospora caninum, Sarcocytis sp. and Eimeria sp. which cause infection in dogs, cattle, poultry, and some mammals, leading to ruin for the livestock industry and other domesticated animals. One of the most infectious Coccidia species that accounts for 30% of global population and can infect virtually any mammalian/bird species is Toxoplasma gondii.

Apicomplexa are unicellular eukaryotes responsible for major human and veterinary diseases such as malaria and toxoplasmosis, which pose massive social and economic burdens. These parasites are defined by a unique polar end known as the apical complex. This complex contains specific secretory machinery required for the invasion of the parasite into host cells (Michael J. Blackman and Bannister, 2001).

They also have a complex lifestyle in which they alternate between sexual and asexual stages in two hosts (dormant vs lytic cycles). These parasites are also identifiable by a non-photosynthetic plastid called the 'apicoplast' which is essential for metabolic processes. Additionally, their mitochondrion genome (mitosome in the case of Cryptosporidida) is highly reduced due to massive gene migrations to the nucleus (Berná, Rego and Francia, 2021).

Plasmodium falciparum

Malaria is arguably one of the most overwhelming infectious diseases throughout the world's existence. It has been documented as early as 2700 BC (Tuteja, 2007). This disease is highly prevalent in poverty-stricken regions of the world such as sub- responsible for the transmission of the parasite between human hosts (Tuteja, 2007).

P. falciparum causes the most severe form of the disease and is the dominant species found in Africa. In rural communities in Africa where proper healthcare is limited the effects of malaria are most noticeable. Around 95% of malaria deaths occur in Africa (World Health Organization, 2021). The spread of drug (artemisinin combine therapy, Nobel 2015 (Youyou, Ōmura and Campbell, 2015)) resistant parasites, the lack of an efficient vaccine, and the inconsistency of malaria control programmes have increased the need for new innovations in Plasmodium research [START_REF] Phillips | Malaria[END_REF].

Toxoplasma gondii

T. gondii is a unicellular eukaryote that infects the cells of warm-blooded animals (mammals and birds), including humans, and causes toxoplasmosis. Toxoplasmosis is a disease considered as asymptomatic in most cases. However, it can be fatal for the foetus in primary-infected pregnant women and for immunocompromised individuals such as AIDS and leukaemia patients (Aparecida Müller De Barros et al., 2022). Despite being well studied, no efficient vaccine exists in preventing transmission of toxoplasmosis.

T. gondii is an excellent model organism for the study of Apicomplexa parasites and intracellular parasitism. The first characteristic that deems this parasite a model organism, is that it can rapidly divide during tachyzoite stages (every 6 to 8 hours) resulting in a high parasite yield within an easily controllably in vitro laboratory system.

This propagation is achieved using several cell-types from warm-blooded animals employing the standard tools of culture cellular. The size of the parasite (five times the size of Plasmodium) makes it easier to observe in optical and electronic microscopy which allows visualization and study of organelles, mechanisms of invasions and the complex structure of the parasite (Kim and Weiss, 2004).

The complex life cycle of apicomplexa parasites

The life cycle of this phylum of parasites is extremely complex and normally consists of two separate host types. This thesis is centred on the two parasites listed above.

The life cycle of haemosporidians and coccidians compose of a primary vector (responsible for the sexual development, gamogony) and a secondary vector (merogony) in which the continuous replication of the parasite will occur.

Plasmodial life cycle

The multistage life cycle of Plasmodial parasites can be separated into three distinct cycles:

The sporogonic cycle (in the mosquito), the exo-erythrocytic cycle and the intraerythrocytic developmental cycle (both in the human host), shown in Figure 1. A human plasmodial infection is initiated when a feeding Anopheles mosquito injects mature sporozoites of the parasite into the bloodstream of the host dermis. This is the beginning of the exo-erythrocytic stage. The sporozoites, transmitted via the saliva of the mosquito, make their way in the bloodstream to the liver of the new host [START_REF] Silvie | Interactions of the malaria parasite and its mammalian host[END_REF]. The initial movement from the dermis to a blood vessel is random and not every sporozoite will reach the hepatocytes in the liver. Those that reach the liver will penetrate the hepatocytes where they remain for 9-16 days and undergo asexual replication known as exo-erythrocytic schizogony (Tuteja, 2007). During this stage a schizont forms within this hepatocyte that is made up of multiple haploid merozoites.

The infected hepatocyte ruptures to release thousands of merozoites into the bloodstream to infect erythrocytes. Asexual division begins inside the erythrocyte, which is also the site for the formation of the other morphological stages of the parasites in the human host. After invasion merozoites develop into ring-stage parasites that will mature to form trophozoites [START_REF] Phillips | Malaria[END_REF]. At the end of the trophic phase, multiple rounds of nuclear division occur through asexual reproduction (erythrocytic schizogony). Schizonts are produced in the red blood cells (RBCs) and consist of up to 32 individual merozoites (Leete and Rubin, 1996). The infected RBCs eventually rupture, releasing the merozoites into the bloodstream where they invade new RBCs. The erythrocytic schizogony process is repeated every 48 hours, (known as the intra-erythrocytic developmental cycle-Figure 1) and coincides with the clinical symptoms of malaria (Tuteja, 2007;[START_REF] Phillips | Malaria[END_REF]. During a blood feed by an infected Anopheles mosquito, sporozoites from their salivary glands enter the blood stream of the mammalian host. The liver stages entail the sporozoites infecting hepatocytes where they then develop in schizont over 48 hours. The hepatic schizont then burst releasing hundreds of merozoites into the blood system of the host which then enter red blood cells. The asexual developmental cycle will now start with the development of ring (24 hours), trophozoites (12-18 hours) and schizonts (6-12 hours) in this sequence over the course of 48 hours. This cycle will continue until proper treatment, a successful immune response or death occurs. The parasites can instead commit to the gametocytogenesis under certain stresses. Stage I-IV gametocytes are found within the blood system of the host, while stage V gametocytes will sequester in the bone marrow until A few of the intra-erythrocytic parasites form male and female gametocytes. These are ingested by the mosquito where the sexual stage of the life cycle takes place. The gametocytes develop into gametes which fuse to form a zygote within the insect's gut.

The zygote develops into an oocyst, where sporozoites are formed and released.

These enter the salivary glands, allowing the parasite to be spread among new human hosts when the mosquito feeds [START_REF] Miller | Malaria biology and disease pathogenesis: insights for new treatments[END_REF].

Toxoplasma life cycle

T. gondii parasites are unique in that they can invade almost any nucleated cell in any warm-blooded host. This contributes to the complexity of their life cycle which is split between two host (intermediate host and definitive host, Figure 2) and three stages depending on their environmental context (Dubey, 2009) :

a) Bradyzoite b) Sporozoite c) Tachyzoite

Final host:

The sexual developmental (Di Genova et al., 2019) stages of this parasite only occur in felines (the definitive host who lacks a desaturase responsible for the formation of linoleic acid). The main source of infection in felines is ingestion of prey containing tissue cysts (bradyzoites) or oocyst containing sporozoites (from other cat faeces).

After ingestion the walls of the cysts are disintegrated and releases bradyzoites or sporozoites after which they invade the epithelial cell making up the wall of the digestion tract. Schizogony of these parasites will give rise to thousands of merozoites that will infect new erythrocytes continuing this lytic component of the cycle. Some may start to differentiate into micro/macrogametes. The fusion of these two sexes leads to the formation of an immature oocysts. As they pass through the intestinal tract, they can be release out of the cat through the cat faeces. The external aerated environment triggers the maturation of the oocysts which are extremely resistant to severe environmental conditions and survive here for months. For further transmission sporozoites ingested by a sequential host to complete its highly lytic-form (tachyzoites) responsible for the spread of the parasite in an intermediate host [START_REF] Attias | The life-cycle of Toxoplasma gondii reviewed using animations[END_REF].

Intermediate hosts:

Intermediate hosts such as humans and cattle ingest sporozoites and bradyzoites from prey/food. Once entering the intestinal tract, these cells can differentiate into tachyzoites. This is a highly proliferative asexual form of the parasite. These stages are characterized by rapid division and dissemination in the host responsible for the symptomatic form of the disease (Dubey, 2009;[START_REF] Attias | The life-cycle of Toxoplasma gondii reviewed using animations[END_REF]. Following rupture of invaded enterocytes, tachyzoites invade monocytes and are spread through to the other cells/organs via the circulatory system (blood). Under certain environmental changes in different tissue (eye, brain, muscle) and immune pressure, tachyzoites can convert to the slow-growing and less-metabolically active bradyzoites stages (as tissue cysts) which are responsible for the chronic phase of toxoplasmosis. These tissue cysts can stay dormant in a host for their entire lifetime and can be re-converted back into tachyzoites under favourable environmental conditions (Francia and Striepen, 2014). This is frequently observed in immuno-deficient patients, chemotherapy and organ recipients which may be fatal in some cases. If a pregnant woman is primoinfected with T. gondii parasites the lack of a memory immune response will allow the transversal of the parasite across the placental membrane and infection of the foetus can lead to congenital conditions or fatality [START_REF] Nhs | Toxoplasmosis[END_REF].

The cellular structure of Apicomplexan parasites

The multi-faceted life cycle of apicomplexan parasites consists of drastic changes to the overall structure of the parasite. This thesis mainly focuses of the rapidly dividing stages namely the merozoite and tachyzoite stages in P. falciparum and T. gondii, respectively. There are some organelles that are shared amongst these forms and contribute to their metabolic similarity. The structure of T. gondii tachyzoites (left) and P. falciparum merozoite (right). ER: endoplasmic reticulum, IMC: inner membrane complex.

The general structure:

Like with all eukaryotic cells, this unicellular parasite (Figure 3) has a nucleus, mitochondria, endoplasmic reticulum (ER, with/without ribosomes) and Golgiapparatus. The ER is largely found at the basal end of the parasite, whereas the Golgi is normally adjacent to the apical end of the parasite nucleus. Many lipid pathways reside within the ER and Golgi, predominantly making up the eukaryotic-type pathway for glycerolipid synthesis (Shunmugam, Arnold, et al., 2022;Walsh et al., 2022). A single mitochondrion exists whose biogenesis is correlated to the cell-cycle [START_REF] Nishi | Organellar dynamics during the cell cycle of Toxoplasma gondii[END_REF]. This intracellular parasite is unique in that it also contains additional membranes once inside the host namely, parasitophorous vacuole membrane (PVM), parasitophorous vacuole (PV), parasite plasma membrane (PM).

The pellicle:

The pellicle is a trilaminar structure which is made up of the outer membrane and the inner membrane complex (IMC, Figure 3). Two continuous membranes make up this cytoskeletal organelle consisting of: sacs of flattened vesicles just beneath the parasite plasma membrane or alveoli and a rigid network of filaments. The IMC is crucial during the development of the parasite and is used as a scaffold in mother parasites during endogeny by daughter cells, which recycling maternal IMC membranes. In T. gondii tachyzoites and bradyzoites, the IMC is composed of three rows of fused rectangular vesicles encircling the parasite with openings at the apical and basal ends (Porchet and Torpier, 1977;[START_REF] Dzierszinski | Dynamics of Toxoplasma gondii differentiation[END_REF]Del Carmen et al., 2009). A similar arrangement is seen in Plasmodium gametocytes where the IMC is formed from 9 to 15 ring-like plates [START_REF] Meszoely | Plasmodium berghei: Architectural Analysis by Freeze-Fracturing of the lntraoocyst Sporozoite's Pellicular System[END_REF][START_REF] Kono | Evolution and Architecture of the Inner Membrane Complex in Asexual and Sexual Stages of the Malaria Parasite[END_REF]. However, in all other Plasmodium life stages, the IMC is formed from a single fused vesicle [START_REF] Dubremetz | Structure de la pellicule du sporozoite de Plasmodium yoelii: étude par cryofracture[END_REF]Bannister and Dluzewski, 1990;[START_REF] Raibaud | Cryofracture Electron Microscopy of the Ookinete Pellicle of Plasmodium gallinaceum Reveals the Existence of Novel Pores in the Alveolar Membranes[END_REF][START_REF] Kono | Evolution and Architecture of the Inner Membrane Complex in Asexual and Sexual Stages of the Malaria Parasite[END_REF].

The apical complex:

The Apicomplexan phylum is defined by the presence of specialised secretory organelles called the apical complex (Cowman, Berry and Baum, 2012) which facilitates secretion and invasion. These organelles include the a) rhoptries (two in P. gondii (Michael J Blackman and Bannister, 2001).

The invasion machinery:

Erythrocytic invasion for Plasmodial is a complex process requiring a sequence if interactions and events to take place (Weiss et al., 2015, Figure 4). The initial contact between the host RBC and merozoite is facilitated by the reversible interaction of the transmembrane erythrocyte band 3 receptor with multiple glycosyl-phosphatidyl inositol (GPI)-anchored merozoite surface proteins [START_REF] Li | A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3[END_REF]. As with Toxoplasma parasites as well, 'gliding motility' facilitates this contact with the new host cells and the apically located organelles (micronemes and rhoptries) act as the main drivers through the secretion of essential proteins. Microneme proteins are initially secreted for irreversible parasite attachment, reorientation, and junction formation such as MIC2 in Toxoplasma and AMA1 in Plasmodium (Narum and Thomas, 1994;Carruthers and Sibley, 1997;Cowman, Berry and Baum, 2012). Thereafter, rhoptries empty their contents (TgRON/ROPs and PfRh/RONs) to enable surface shedding, actin-myosin motility, and eventual vacuole formation to all the cell to be internalized by its new host (Cowman, Berry and Baum, 2012). Finally, post-invasion after PV formation, the dense granules proteins, called GRA proteins are released into the PV and further excreted out into the host nucleus (Hakimi and Bougdour, 2015;Mercier and Cesbron-Delauw, 2015). GRA proteins play a critical role in the interaction with the host as they can adapt the host cell gene expression depending on the parasite cell requirements.

The metabolic organelles:

Apicomplexan parasites possess a single apicoplast and mitochondrion like their algal ancestors (Figure 5).

Apicoplast:

A primary endosymbiotic event which, involved the engulfment of Cyanobacterium by a eukaryotic heterotroph gave rise to the chloroplast we find in plants and algae. This organelle was acquired by several apicomplexan in an event involving secondary endosymbiosis where plastids of Chromalveolata were derived from red algae (Lim and McFadden, 2010;van Dooren and Striepen, 2013). Their coevolution during the ancestral acquisition of this relict plastid and subsequent loss of photosynthesis resulted in divergent metabolic pathways compared to mammals and plants. Due to its evolutionary origins the apicoplast is made up of four membranes enriched in phospholipids and not galactolipids like their algal ancestors (Botté et al., 2013). This organelle houses 4 principle metabolic processes (Figure 6) which are crucial to the development of the parasite and can be considered the parasites' kryptonite: a) FASII pathway: An apicoplast-located fatty acid synthesis is dispensable in erythrocyte stages of the malaria parasite, which instead predominantly salvage fatty acids. However, the indispensability of the FASII pathway has been proven in Plasmodium liver stages and Toxoplasma tachyzoites. The total contribution of short chain fatty acids (palmitate, myristate) required for membrane biogenesis is attributed to the apicoplast FASII. This pathway aids in parasite division and survival within the host (Mazumdar and Striepen, 2007;Ramakrishnan, Melissa D Docampo, et al., 2012a;Amiar et al., 2016;Amiar, Nicholas J Katris, et al., 2020a).

b)

Isoprenoid precursor assembly pathway: Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle and are important lipid components involved in tRNA modification, protein prenylation, ubiquitination etc.

There are two pathways that contribute to their synthesis: a eukaryotic mevalonate pathway and a prokaryotic MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate) pathway. Isoprenoid precursor synthesis is the only essential role of the apicoplast in Plasmodium erythrocytic stages [START_REF] Baumeister | Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways[END_REF]Yeh and Derisi, 2011). Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols [START_REF] Okada | Critical role for isoprenoids in apicoplast biogenesis by malaria parasites[END_REF]. The DOXP pathway is also essential in Toxoplasma but a lack of sensitivity to drugs targeting this pathway in tachyzoites suggests the pathway cannot be exploited due to delivery across host and parasite membranes [START_REF] Nair | Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii[END_REF]Sheiner, Vaidya and Mcfadden, 2013). c) Iron (Fe)-sulphur cluster biosynthesis: These clusters are a common prosthetic group essential for protein function by mediating electron binding and transport, deeming them critical for redox reaction in the cell. Of the 4 Fe-S cluster synthesis pathways only one gene of the Suf pathway is encoded in the apicoplast genome [START_REF] Waller | Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum[END_REF]Salomaki and Kolisko, 2019). This pathway supplies Fe-S clusters for enzymes producing isopentenyl pyrophosohate (IPP), which is required for the protein/pathway preventing the loss of the apicoplast (Ralph, van Dooren, et al., 2004).

Retention of the apicoplast genome seems to be strongly correlated to the Suf pathway.

d)

Heme biosynthesis pathway: Heme is a prosthetic group added to many proteins to allow them to carry small molecules/electrons for many redox reactions.

This process takes place the apicoplast, mitochondria and the cytosol. Only one homologue of the process has been retained in the apicoplast of these parasites. The other steps have been transferred to the other cellular compartments. Heme synthesis is essential for liver and mosquito stages of Plasmodium parasites. In RBC stages, the parasite is in an environment of excessive heme levels and instead it is processed and stored in the form of hemozoin to prevent toxicity [START_REF] Kořeny | Tetrapyrrole Synthesis of Photosynthetic Chromerids Is Likely Homologous to the Unusual Pathway of Apicomplexan Parasites C W[END_REF]Salomaki and Kolisko, 2019). Recent work has shown the evidence of a function heme biosynthesis pathway in the Toxoplasma apicoplast [START_REF] Bergmann | Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection[END_REF].

Mitochondria:

The mitochondrial genome only encodes for 3 proteins and instead mitochondrial genes are transferred to the nucleus to allow for nuclear control. This organelle is in close physical proximity to the apicoplast and essential for both Toxoplasma and Plasmodium spp., being the synthesis 'factory' for several metabolites. The Tricarboxylic acid cycle (TCA, Figure 7) is central to mitochondrial metabolism and contributes to biosynthetic and catabolic functions as being the primary source of electrons that fuel, the mitochondrial electron chain generating ATP. The 'textbook'

TCA cycle is fed acetyl-CoA from the production of pyruvate from glycolysis, the breakdown of branched amino acids or the β-oxidation of fatty acids (FA). Acetyl-CoA is combined with oxaloacetate to form citrate, which then enters a multi-stepped process which results in CO2, NADH, FADH2 and GTP, and ultimately regenerates oxaloacetate (van Dooren, Stimmler and Mcfadden, 2006;Seeber, Limenitakis and Soldati-Favre, 2008;Sheiner, Vaidya and Mcfadden, 2013). The exact processes contributing to the acetyl-CoA pool for the TCA cycle is not entirely known. Only one pyruvate dehydrogenase (PDH) complex is present in the apicoplast which seems not to appreciably contribute to acetyl-CoA pool [START_REF] Foth | The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast[END_REF][START_REF] Crawford | Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast[END_REF][START_REF] Fleige | Carbohydrate metabolism in the Toxoplasma gondii apicoplast: Localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator[END_REF]. Instead, evidence points towards a branched-chain keto acid dehydrogenase (BCKDH) complex being the missing link required for fuelling the TCA cycle [START_REF] Macrae | Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum[END_REF][START_REF] Oppenheim | BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei[END_REF][START_REF] Kloehn | Multi-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii[END_REF] in all apicomplexa (except Cryptosporidium). Sheiner, Vaidya and Mcfadden, 2013;Demas, 2016;Moog, Przyborski and Maier, 2017, Oppenheim et al., 2014and Nugraha, Jeelani and Nozaki, 2022.

Peroxisome:

Peroxisomes are multifunctional organelles present in virtually all eukaryotic cells.

They are extremely plastic and can transform their shape, size (0.1-0.5µM diameter) number and function strictly depending on their external environment (Lodhi and Semenkovich, 2014). These single membrane organelles normally house several oxidases and catalases involved in hydrogen peroxide metabolism and are tightly associated to fatty acid oxidation. This organelle is unique in that it is not essential under standard nutritional conditions, i.e. in the presence of favoured carbon sources such as glucose. In yeast, it is known that excessive oleic acid induces peroxisome formation and the upregulation of peroxisomal proteins (Kohlwein, Veenhuis and van der Klei, 2013). Interestingly, in yeast β-oxidation solely occurs in the peroxisomes and is completely absent of mitochondrial β-oxidation which is preferred in most other eukaryotes (Figure 7).

Peroxisomal biology has only recently been investigated in the field of apicomplexan parasites and now bioinformatical and wet-lab studies suggest (with some debate) the existence of a peroxisome (Kaasch and Joiner, 2000;[START_REF] Ludewig-Klingner | Distribution and Evolution of Peroxisomes in Alveolates (Apicomplexa, Dinoflagellates, Ciliates)[END_REF]Moog, Przyborski and Maier, 2017). Microscopy studies have hinted at singlemembrane peroxisomes in P. falciparum parasites (Mcintosh, Elliott and Joiner, 2005) but it seems they lack some proteins traditionally involved in β-oxidation. Under starvation conditions, FA oxidation metabolism has been demonstrated in P.

falciparum parasites despite not being previously annotated (Demas, 2016).

No structural or metabolic proof illustrates the presence of peroxisomes in Toxoplasma parasites. Genomic and proteomic studies now show the existence of peroxisomes in this parasite (Moog, Przyborski and Maier, 2017). For this thesis, it is of interest to acknowledge the loss of a carnitine palmitoyl acyltransferase in Toxoplasma, required for the transport of acyl-CoA across the mitochondrial membrane for fatty acid βoxidation [START_REF] Danne | Alveolate Mitochondrial Metabolic Evolution: Dinoflagellates Force Reassessment of the Role of Parasitism as a Driver of Change in Apicomplexans[END_REF]. It is likely that instead a peroxisomal-like organelles exists as all fatty-acid oxidation are present in Toxoplasma, but in Plasmodium spp., the functions of this organelle are fulfilled by scavenging by-products from the host (Ding, Clayton and Soldati, 2000;Kaasch and Joiner, 2000;[START_REF] Mbekeani | Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii[END_REF].

Lipid droplets:

Lipid droplets (LDs) also known as lipid bodies/lipid particles/oil bodies, like peroxisomes, are considered highly dynamic organelles heavily dependent on the cell nutrient status. This single-membraned organelle is known as the inert lipid storage depots/banks primarily comprised of triacylglycerol (TAG) or sterol esters used under sub-optimal conditions (Kohlwein, Veenhuis and Van Der Klei, 2013;[START_REF] Vallochi | Lipid Droplet, a Key Player in Host-Parasite Interactions[END_REF]. LDs have been observed in asexual trophozoite stage P. falciparum parasites (one per parasite) but seem to be absent in gametocyte stages [START_REF] Jackson | Food vacuoleassociated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum[END_REF]Gulati et al., 2015). LDs are closely associated to the food vacuole where heme is converted to hemozoin at it is believed that the internalized host phospholipids (PLs) are converted to neutral lipids (TAG sterol esters) and redirected to LDs [START_REF] Jackson | Food vacuole-associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum[END_REF]. Gulati et al., (2015) showed that inhibition of TAG-mobilization from LD prompted gametocyte conversion.

Intracellular Toxoplasma tachyzoites recruit host LDs and mitochondria to facilitate the scavenging of certain biomolecules that are not de novo synthesized (i.e. Cholesterol) (Charron and Sibley, 2002;Nolan, Romano and Coppens, 2017a) or deficient (low DAG/TAG levels) (Shunmugam, Arnold, et al., 2022). The knockdown of a phosphatase (TgLIPIN) illustrates that the channelling between active membrane biogenesis and the storage of lipid in the form of LDs is crucial to the survival of the parasite and prevents lipotoxic death (Shunmugam et al., 2021).

In other eukaryotic organisms LDs and peroxisomes are in proximity within the cell (Lodhi and Semenkovich, 2014) and is thought to be metabolically driven depending on the external environment. Whether there is intimate crosstalk between these two organelles in Apicomplexa parasites is yet to be fully investigated.

Lipids constitute one of the fundamental building blocks of life and apicomplexan parasites are no different in demanding this crucial resource through de novo synthesis within the apicoplast (FASII) and host/environment scavenging. Lipid metabolism is critical to signalling, energy storage and membrane biogenesis. Their importance in several cellular functions renders their pathways as potential drug targets (Coppens and Botté, 2020). The enzymes of the apicoplast-derived fatty acid synthesis pathway have already been successfully exploited as a target against Toxoplasmosis infections and heavily studied in P. falciparum [START_REF] Mcleod | Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I[END_REF][START_REF] Perozzo | Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase[END_REF][START_REF] Surolia | FAS't inhibition of malaria[END_REF]Botte et al., 2011). There are multiple classes of lipids that must be considered (Shunmugam et al., 2022).

Fatty acids (FA) and activation:

Most lipids contain fatty-acyl chains as part of their structure (De Carvalho and Caramujo, 2018). These biomolecules have numerous functions including structural elements of phospholipid making up the membranes of the cell, serving as a part of neutral lipids for energy storage and derivatives being implicated in cell signalling.

These parasites are unique in that they can source these FA from the host cell (FASIfatty acid synthase I), host environment or synthesize them de novo by the FASII in the apicoplast. The central accepted dogma in Apicomplexan parasites is that then they can compile a patchwork assembly of lipids from these different sources (Shunmugam et al., 2022).

Prior to the use of these FA in anabolic and catabolic pathways, they must be metabolically activated via the thioesterification with coenzyme A, coupled with adenosine-triphosphate (ATP) molecule. This two-step reaction is facilitated by a group of enzymes called acyl-CoA synthetases (ACSs, Figure 7) (Trisha J. Grevengoed, Klett and Coleman, 2014). Once activated these fatty acyl-CoA molecules can participate in:

• transcriptional regulation

• phospholipid (PL) and ceramide synthesis (via Kennedy/lands pathway)

• neutral lipid synthesis (di/triacylglycerol, cholesterol, esters)

• protein acylation

• FA elongation, desaturation • FA degradation (β/ω-oxidation)
In most eukaryotic models 4-6 genes encode for ACSs, but 11 and 7 putative genes exist in P. falciparum and Toxoplasma parasite (Bethke et al., 2006;Shunmugam, Dass, et al., unpublished-see Chapter 4) is involved in neutral lipid metabolism under sub-optimal nutrient conditions). These proteins seem to localize to different parts of the parasite suggesting non-redundant functions. Once these FA are activated, they can contribute to the formation of the lipid groups described further.

Phosphatidylcholine (PC):

One of the most predominant phospholipids (PLs) in all cells is phosphatidylcholine (PC) since this is a major contributor to cell membranes. This is no different in apicomplexan parasites as the relative abundance of PC is 40-70% for T. gondii and 40-50% for P. falciparum parasites [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF][START_REF] Welti | Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids[END_REF]Gulati et al., 2015). The deciding group of this class of lipids, choline, is derived from the external environment of T. gondii tachyzoites. A cytosolic choline-kinase facilitates the de novo Kennedy pathway which results in the essential synthesis of PC [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF]Sampels et al., 2012, Figure 8). A nuclear CTP: phosphocholine cytidylyltransferase (PfCCT) in P. falciparum is the second rate-limiting step of the de novo synthesis of PC. Phospholipase C treatment induces translocation of this protein to the cytosol along with an increase PC biosynthesis [START_REF] Izrael | Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme[END_REF]. Malaria parasites have also adapted a different strategy by producing PC via a pathway called decarboxylation-phosphoethanolamine methylation (SDPM) that uses host ethanolamine and serine (Pessi, Kociubinski and Ben Mamoun, 2004;[START_REF] Pessi | In Vivo Evidence for the Specificity of Plasmodium falciparum Phosphoethanolamine Methyltransferase and Its Coupling to the Kennedy Pathway*[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]. In phospholipase, PfLPL20, depleted parasites, transcript levels of enzymes in the Serine Decarboxylase-Phosphoethanolamine

Methyltransferase pathway (SDPM) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway (P. Sheokand et al., 2021). In contrast Toxoplasma cannot use these as substrates to produce PC since parasites lack the required methyltransferase activity [START_REF] Gupta | Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth[END_REF].

Phospholipases govern the balance between PC and lyso-PC (LPC). High LPC levels in malaria parasite repress RBC stage sexual conversion (Brancucci et al., 2017). A TgPL2 (phospholipase 2) was identified that is responsible for the hydrolysis of a FA from PC to form LPC. The increase in PC when this protein is disrupted is combined with a destruction of the apicoplast membrane integrity, suggesting LPC/PC levels are important in maintaining the apicoplast homeostasis (Lévêque et al., 2017). acyltransferase: AT, diacylglycerol: DAG. This figure has been adapted from [START_REF] Ramakrishnan | Lipid Synthesis in Protozoan Parasites: a Comparison Between Kinetoplastids and Apicomplexans[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]Flammersfeld et al., 2017;Kilian et al., 2018;[START_REF] Neveu | Metabolic regulation of sexual commitment in Plasmodium falciparum[END_REF]

Phosphotidylethonalmine (PE):

This phospholipid is the second most abundant in these parasites with 10-20% for T.

gondii and 15-25% for P. falciparum parasite [START_REF] Gupta | The Obligate Intracellular Parasite Toxoplasma gondii Secretes a Soluble Phosphatidylserine Decarboxylase *[END_REF]Gulati et al., 2015).

PE in Toxoplasma is made via a mitochondrial phosphatidylserine decarboxylase (TgPSD, Figure 8) or ER fusion of CDP-ethanolamine and diacylglycerol (DAG). The loss of TgPSD leads to suboptimal growth of the parasite but is rescued by exogenous ethanolamine [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]. PE synthesis in P. falciparum is multifaceted and uses the CDP-ethanolamine/Kennedy pathway as well as a serine decarboxylase pathway (Elabbadi, Ancelin and Vial, 1997;Kilian et al., 2018). PE can also be derived from PC, the inhibition of a PfCholine kinase (PfCK) leads to a decrease in PE and ultimately parasite death [START_REF] Serrán-Aguilera | Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death[END_REF].

Phosphatidylserine (PS):

This phospholipid is somewhat abundant as it comprises only 5-10% of the relative abundance of lipids in P. falciparum and Toxoplasma parasites. This lipid is exclusively localized to the inner leaflet of the lipid bilayer of cell membranes. However, during certain stresses in eukaryotes PS can flip onto the surface of cells acting as a global immunosuppressor by mimicking apoptosis to evade host immune responses [START_REF] Alberts | Molecular Biology of the cell[END_REF][START_REF] Birge | Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer[END_REF]. Exposure of PS on the surface of malaria-infected red blood cells contribute to their cytoadherence/sequestration and disease pathogenesis in P. falciparum malaria (Eda and Sherman, 2002). This is supported by increased levels of PS in P. falciparum positive patients (Gulati et al., 2015).

Unlike Plasmodium that has a single protein (PfPSS), in Toxoplasma there are two enzymes responsible for the formation of PS (using serine as a substrate) near the ER;

TgPSS (phosphatidylserine synthase) and TgPTS (phosphatidylthreonine synthase) (Arroyo-Olarte et al., 2015, Figure 8). Malarial parasites can use host-derived PS through the degradation of host haemoglobin (Wein et al., 2018). PE can also be used as a precursor of PS synthesis via serine decarboxylation path in both these parasites (Elabbadi, Ancelin and Vial, 1997;Kilian et al., 2018).

Phosphatidylthreonine (PT):

Phosphatidylthreonine (PT) is unique in Toxoplasma as it is a major phospholipid facilitating lipid mediated virulence in the host [START_REF] Arroyo-Olarte | Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence[END_REF]. TgPTS, a protein which has evolved from TgPSS (since they are both capable of PS synthesis), is used to generate PT within the ER (Figure 8).

It appears that PS and PT synthesis is a fine balance in this parasite as genetic disruption of TgPTS resulted in increased levels of PS and correlated decrease in PT (more specifically PT species 20:4 and 20:1 which are host-derived) [START_REF] Arroyo-Olarte | Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence[END_REF]. This balance influences the calcium flux and storage controlling parasite egress (Arroyo-Olarte and Gupta, 2016;[START_REF] Kuchipudi | Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii[END_REF]. Very little is known about the presence and role, if any, of PT in Plasmodium (PlasmoDB, 2022).

Phosphatidylinositol (PI) and related phosphoinositides

Pl, which is negatively charged, accounts for 5% of the total PL composition and has critical functions such and intracellular trafficking and cell signalling. A phosphatidyl-3monophosphate kinase in Toxoplasma parasites is involved in apicoplast biogenesis and lipid trafficking to and from said-organelle [START_REF] Tawk | Phosphatidylinositol 3-Monophosphate Is Involved in Toxoplasma Apicoplast Biogenesis[END_REF]. Another protein, TgPH1, binds to the phosphoinositide PI (3,5)P2 and serves a complementary reporter of this molecule in mammalian cells [START_REF] Daher | Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P2[END_REF][START_REF] Chintaluri | The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells[END_REF]. A phospholipase, TgPI-PLC, has a high affinity for PI and localises to the plasma membrane and cytosol of the parasite suggesting plural roles in lipid regulation. (Fang, Marchesini and Moreno, 2006;Bullen et al., 2016).

A phosphoinositide-3-kinase (PI3K) in P. falciparum is actively exported into the host RBC and allows for the endocytic trafficking of toxic haemoglobin from the host to the parasite food vacuole [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]. A prominent drug discovery against malaria is MMV390048, an inhibitor of phosphatidylinositol 4-kinase [START_REF] Paquet | Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase[END_REF][START_REF] Fienberg | Structural Basis for Inhibitor Potency and Selectivity of Plasmodium falciparum Phosphatidylinositol 4-Kinase Inhibitors[END_REF]. The resulting product of this enzyme is a sugar-derivative of PI like glycosylphosphatidylinositol (GPI), which is known as a major pathogenicity determinant of the parasite being able to induce TNF-α (tumor necrosis factor alpha) production [START_REF] Debierre-Grockiego | Fatty Acids from Plasmodium falciparum Down-Regulate the Toxic Activity of Malaria Glycosylphosphatidylinositols[END_REF]. This parasite directly channels hostscavenged myo-inositol for GPI synthesis, but uses a combination de novo synthesized, and host myo-inositol for other PI derivatives [START_REF] Macrae | Plasmodium falciparum is dependent on de novo myo-inositol biosynthesis for assembly of GPI glycolipids and infectivity[END_REF].

Phosphatidylglycerol (PG):

Gulati et al. (2015), show that phosphatidyl-glycerol only accounts for 2-3% of the total PL content of P. falciparum parasites. This anionic PL increases from undetectable to 4% in infected RBC cells (Tran et al., 2016), and decreases to 1% under gametocyte maturation (Figure 9). In other organisms, this PL is also involved in lysobisphosphatidic acids acid (LBPA, or bis (mono-acylglycerol) phosphate (BMP))

and lysophosphatidylglycerol (LPG) formation though the activity of a phospholipase A2 protein that hydrolyses a FA (Flammersfeld et al., 2017). A stage-dependence dynamic of LPG abundance has been shown in malaria parasites [START_REF] Tewari | Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite[END_REF].

LPG is believed to be involved in calcium-cell signalling (Zhang et al., 2017). Both LPG and LBPA levels increase 2/3-fold in P. falciparum infected RBCs (Gulati et al., 2015;[START_REF] Tewari | Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite[END_REF]. It is hypothesized that LBPA, which spike after 48 h post-infection is related to the formation of vesicles and membrane fusion for merozoite cell remodelling. LBPA is enriched in late endosome-lysosome compartment crucial for endocytosis (Gruenberg, 2020). Human macrophages lacking Niemann-Pick C genes (displaying an autosomal disorder), lead to a disruption of endosomal lipid transport proteins and an accumulation of cholesterol, which can be rescued by exogenous PG resulting in LBPA production leading to endosomal clearing of cholesterol [START_REF] Ilnytska | Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage[END_REF]. This highlights the importance of the PG/LBPA balance.

PG synthases (from cytidine diphosphate-diacylglycerol) have not been identified in either parasite, but genes encoding homologous proteins (PF3D7_0820200 and TGVEG_005460) do exist (Ramakrishnan et al., 2013;PlasmoDB, 2022;VEuPathDB, 2022). Not much has been revealed about the role of PG in Toxoplasma, besides that it is essential for cardiolipin (CL) synthesis and mitochondrial homeostasis (Ramakrishnan et al., 2013). (Gulati et al., 2015;[START_REF] Tewari | Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite[END_REF]

Cardiolipin

Under plasmodial infections, cardiolipin (CL) levels and anti-CL antibodies are elevated in patients, again emphasizes that lipids are very important for the virulence of parasites [START_REF] Jakobsen | Anti-phospholipid antibodies in patients with Plasmodiumfalciparum malaria[END_REF][START_REF] Consigny | High prevalence of co-factor independent anticardiolipin antibodies in malaria exposed individuals[END_REF]. CL in an anionic dimeric phospholipid which usually localizes to the mitochondrial inner membrane of eukaryotic cells. It is closely associated with mitochondrial function, biogenesis, and genome stability (Mai and Altucci, 2009). CL is synthesized from phosphatidyl-CMP and phosphatidylglycerol (PG) (Schlame, 2008). Studies now show that CL induces the activation and transport of mitochondrial P. falciparum proteins [START_REF] Nozawa | Characterization of mitochondrial carrier proteins of malaria parasite Plasmodium falciparum based on in vitro translation and reconstitution[END_REF].

A cardiolipin synthase (CLS) has identified but not investigated in Plasmodium (PF3D7_0609400) (Ramakrishnan et al., 2013).

Similarly, to Plasmodium, Toxoplasma parasites have a common mechanism in making CL from PG (TGGT_309940) de novo. The fatty acid transport, acyl-CoA binding protein 2, TgACBP2 is crucial for CL synthesis and consequently sustains mitochondrial homeostasis. A mitochondrial association factor (MAF1) mediated host mitochondrial association in host cells can restore the growth of TgACBP2-deficient cells, again highlighting the importance of plural-source nature of lipids like CL in this case. Additionally, cryo-EM experiments have shown that a mitochondrial ATP synthase, playing a critical role in the curvature of mitochondrial cristae in the parasite, forms a heteromer bound to CL molecules, suggesting this lipid plays a role on energy conversion [START_REF] Mühleip | ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria[END_REF].

Cytidine diphosphate-diacylglycerol (CDP):

Cytidine diphosphate-diacylglycerol (CDP) is a nucleotide that is at a central branchpoint of lipid metabolism. CDP synthases (CDS), the protein responsible CDP production, is often considered a gateway enzyme into the synthesis of PI and CL.

CDS can regulate PA synthesis and subsequently intracellular PA signalling (Jennings and Epand, 2020). PA levels can also influence membrane curvature and hence membrane trafficking between organelles. Toxoplasma parasites house two distinct CDS isoforms; a eukaryotic-type TgCDS1 found in the ER and a prokaryotic-type TgCDS2 found in the apicoplast. Deletion of the respective proteins showed decreases in PI and PG levels, and more importantly reduced the CDP-DAG pools in the Golgi apparatus and mitochondrion. Overall, when the CDP-DAG pathway was affected, parasite virulence in mice was attenuated [START_REF] Kong | Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii[END_REF]. There is only a single P.

falciparum homolog of CDS that is proteolytically cleaved. Deletion of the N-terminal end of this protein was detrimental to parasite survival [START_REF] Shastri | Plasmodium CDP-DAG synthase: An atypical gene with an essential Nterminal extension[END_REF].

Sphingomyelin:

Sphingolipids de novo synthesis takes places within the ER-Golgi apparatus network.

This class of lipids is defined by their 18-carbon amino-alcohol backbones (Adada, Luberto and Canals, 2016). Sphingomyelin synthase (SMS) is responsible for transferring the phosphorylcholine group from PC to ceramide, eventually resulting in DAG and sphingomyelin. Thereafter, this lipid is transported to other organelles for a diverse array of signal transduction pathways (Gault, Obeid and Hannun, 2010).

These parasites have retained a ceramide biosynthesis pathway via two ER-confined ceramide synthases (TgCERS1/2) with only one having ceramide synthase activity but both being indispensable for parasite fitness [START_REF] Koutsogiannis | Toxoplasma ceramide synthases: a curious case of gene duplication, divergence and key functionality[END_REF]. T. gondii parasites caenge sphigomyelins but synthesis their own ceramide phosphoethanolamines (EPC). Recent work now show that sphingolipid synthesis affects the chronic infection in vivo [START_REF] Nyonda | Ceramide biosynthesis is critical for establishment of the intracellular niche of Toxoplasma gondii[END_REF].

Toxoplasma sphingolipids account only for 1% of the total lipid abundance, but contrastingly has an unusually high level or EPC [START_REF] Welti | Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids[END_REF]. A sphingolipid synthase (TgSLS) has been biochemically validated as an inositol phosphorylceramide (IPC) synthase [START_REF] Pratt | Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii[END_REF].

Metabolic pulse-chase experiments have showed that this parasite scavenges serine and galactose for de novo glycosphingolipid synthesis [START_REF] Azzouz | Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii[END_REF]. This parasite is also highly dependent on scavenging of sphingolipids from the host. Rabcoated vesicles facilitate the recruitment of sphingolipids to the vacuolar spaces surrounding the parasite. When the sphingolipid metabolism of host cells is inhibited, by myriocin, tachyzoite replication is decreased suggesting that they still strongly rely on host-scavenging 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].

In P. falciparum parasites, the asexual stages have an abundance of 10-15% of sphingomyelin, being the second most abundant class of lipids in this stage [START_REF] Gulati | Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum[END_REF]. There is also the unusual presence of dihydrosphingomyelin (dhSM). An extensive tubovesicular membranes (TVM) extending from the PVM into the host cell is necessary for nutrient uptake [START_REF] Lauer | A Membrane Network for Nutrient Import in Red Cells Infected with the Malaria Parasite[END_REF]. The parasite is also able to export sphingomyelin into host cells to form these TVMs [START_REF] Elmendorf | Plasmodium falciparum Exports the Golgi Marker Sphingomyelin Synthase into a Tubovesicular Network in the Cytoplasm of Mature Erythrocytes[END_REF]Van Wye et al., 1996). Sphingomyelin synthase (SMS) activity has been seen in the Golgiapparatus (Lauer, Ghori and Haldar, 1995). Recent work identifies two PfSMS that contribute to gametocytogenesis and sex-specificity. They also show dhSM enrichment in female gametocytes and suggest that this lipid is crucial for gametocyte viability [START_REF] Ridgway | Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole[END_REF].

Cholesterol and cholesteryl esters:

This sterol is largely hydrophobic, localizes in eukaryotes to the plasma membrane where it is often packed with sphingolipids and GPI-anchored proteins establishing roles in the regulation of membrane trafficking, signalling and host-pathogen interactions. They are also concentrated within LD as esterified forms, i.e. cholesteryl esters (Luo, Yang and Song, 2020). Cholesterol is located within the PM and apical organelles of tachyzoites (Coppens, Sinai and Joiner, 2000).

Toxoplasma and P. falciparum parasites are auxotrophic for cholesterol and require scavenging them from the host cell and environment. Numerous studies have shown that host cholesterol levels impact T. gondii parasite replication [START_REF] Bottova | Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii[END_REF]Hu, Binns and Reese, 2017;Nolan, Romano and Coppens, 2017a). More recently, it is now suspected that this lipid plays a role on the modulation of antigen presentation [START_REF] Croce | Efficient Cholesterol Transport in Dendritic Cells Defines Optimal Exogenous Antigen Presentation and Toxoplasma gondii Proliferation[END_REF]. This parasite exploits host low-density lipoproteins (LDL) to direct exogenous cholesterol. A host membrane bound P-glycoprotein acts as an efflux pump and participates in cholesterol export towards the parasite [START_REF] Bottova | Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii[END_REF].

Toxoplasma can make cholesteryl esters from host-derived cholesterol. A lecithin:cholesterol acyltransferase (LCAT) has been identified in Toxoplasma , a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues [START_REF] Pszenny | A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress *[END_REF]. Additionally, this parasite houses two acyl-CoA:cholesterol acyltransferase enzymes (ACAT) directly involved in channelling cholesterol into LD (Lige, Sampels and Coppens, 2013). Deletion of any of these ACAT enzymes leads to either a reduction in virulence/egress or parasite death due to an injurious over-production of membranes [START_REF] Nishikawa | Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma[END_REF][START_REF] Pszenny | A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress *[END_REF]Schultz and Carruthers, 2018).

Plasmodium parasites do not produce any sterols and uses the human mevalonate pathway to generate IPP for sterol/sterol ester synthesis with via LDLs/HDLs as carriers [START_REF] Grellier | Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells[END_REF]Coppens and Vielemeyer, 2005;[START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF].

PfNiemann-Pick Type C1-Related protein (PfNCR1) is suspected to be involved in this import of cholesterol, which is governed a sodium and cholesterol gradient in infected RBC found across the host membrane, PVM and PPM [START_REF] Istvan | Plasmodium niemann-pick type C1-related protein is a druggable target required for parasite membrane homeostasis[END_REF][START_REF] Hayakawa | Real-time cholesterol sorting in Plasmodium falciparum -erythrocytes as revealed by 3D label-free imaging[END_REF][START_REF] Ahiya | Dramatic Consequences of Reducing Erythrocyte Membrane Cholesterol on Plasmodium falciparum[END_REF]. In RBC stage parasites a cholesterol transport system exists, whereby free RBC-membrane derived cholesterol is directed into the cytoplasm of the parasite via the intrusion of elongated lipid membranes that eventually fuse with the PVM (H. Hayakawa et al., 2020). RBC cholesterol levels decrease upon parasite infection, but gametocyte formation leads to a relative increase in RBC-cholesterol (Tran et al., 2016). Cholesterol is not stored in the form of free cholesteryl esters and is suspected to be toxic to parasites when levels are high, hence their incorporation in LD [START_REF] Tran | A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite[END_REF].

Phosphatidic acid (PA):

Phosphatidic acid (PA) is the simplest glycerophospholipid (negatively charged) and is considered a central precursor to all other glycerophospholipids and neutral lipids like diacylglycerol (DAG) and triacylglycerol (TAG). This lipid plays a crucial role in membrane curvature (cone shaped symmetry), thereby allowing the remodelling of the membrane for key protein interaction or events like fission and fusion (Bullen and Soldati-Favre, 2016;[START_REF] Tanguy | Phosphatidic acid: From pleiotropic functions to neuronal pathology[END_REF]. Despite PA's importance, it only accounts of 1% in Toxoplasma and P. falciparum asexual parasites [START_REF] Welti | Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids[END_REF][START_REF] Gulati | Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum[END_REF]. An acylglycerol-3-phosphate acyltransferase (AGPAT) can form PA through the esterification of a second FA on the sn-2 position of the LPA. PA can also be formed via the phosphorylation of DAG via a DAG-kinase (DGK) (Bullen and Soldati-Favre, 2016;Bullen et al., 2016;Shunmugam, Arnold, et al., 2022;Walsh et al., 2022).

A phosphatase, TgLIPIN, is responsible for the conversion of PA to DAG which is essential in preventing 'lipotoxic' death by channelling host-derived FAs to LD molecules (DAG/TAG) and phospholipid membrane biogenesis (Shunmugam et al., 2021). This work made up a significant part of my thesis work that was started by a previous student, Dr Sheena Dass.

PA is key for parasite biology and lipid metabolism contributing to lipid signalling (Bullen et al., 2016), egress/invasion (Bisio et al., 2019a), motility, cytokinesis/division (Amiar, Nicholas J Katris, et al., 2020a) and ultimately what we discovered: maintaining a fine balance between lipid storage and membrane biogenesis (Shunmugam et al., 2021).

Diacylglycerol and triacylglycerol (DAG/TAG):

DAG and TAG are essential neutral lipids found highly concentrated in LDs. They are extremely hydrophobic and energetically dense. The FAs esterified onto these molecules can be hydrolysed and channelled toward energy metabolism via βoxidation.

DAG is implicated in key signalling events (Bullen and Soldati-Favre, 2016). Bullen et al., showed that TgDGK1 regulates microneme secretion through a PA-mediated reaction with a microneme surface protein (Bullen et al., 2016). PA acts as a lipid regulator for microneme exocytosis. DGK2 is the second kinase of this kind that is secreted into the PV and leads to PA formation from DAG, which acts as an intrinsic signal eliciting egress upstream a guanylate cyclase (GC) unique to alveolates (Bisio et al., 2019a).

DAG also play a crucial role as the substrate of TAG synthesis. Two diacylglycerol acyltransferases (DGAT) exist in Toxoplasma (Quittnat et al., 2004;[START_REF] Toxodb | ToxoDB[END_REF];

TgDGAT1 and TgDGAT2. TgDGAT1 localizes to the parasite ER and favourably incorporates C16:0 in TAG moieties. Oleic acid (C18:1) is the predominant FA species found in TAG of LD. Exogenous oleic acid of Toxoplasma parasites lead to an increase in the transcription of TgDGAT1, suggesting a nutrient dependant role of this protein.

Additionally excessive oleic acid (more than 0.5 mM) a lipotoxic retort was provoked, which led to parasite death (Nolan, Romano and Coppens, 2017b). Parasites treated with a known DGAT inhibitor (T863), caused ER-membranous structures which can be further exaggerated with the addition of high oleic acid levels in tachyzoites. In bradyzoites this treatment reduced LD and led to poorly formed cysts (Nolan et al., 2018). Our work (Shunmugam et al., 2021) • A fine equilibrium between lipid acquisition, membrane biogenesis and lipid storage must be maintained to promote parasite progression.

•

The nutrient environment is highly variable for these parasites depending on the cells they infect and the life stages. This means that they must be able to maintain this balance while being extremely metabolically plastic.

Résumé du travail publié (Shunmugam, Arnold, et al., 2022):

• Les lipides sont essentiels à la survie des parasites et particulièrement au cours des phases de division rapide comme la phase sanguine de P. falciparum et T.

gondii au stade tachyzoite.

• • Les mécanismes de capture de lipides d'origine exogène diffèrent entre les parasites Toxoplasma et Plasmodium, et en fonction de leurs stades de développement. Ces mécanismes sont décrits dans cette revue.

• Un équilibre doit être maintenu entre l'acquisition de lipide, la synthèse de membrane et le stockage de lipides pour permettre la progression du parasite.

• L'environnement nutritif est très variable pour ces parasites en fonction du type de cellules qu'ils infectent et de leur stade de développement. Ils doivent donc être capables de maintenir cet équilibre en adoptant des stratégies de plasticité métabolique. Les résultats principaux sont les suivants :

• TgLIPIN, la seule LIPIN présente dans le génome de T.gondii, est une protéine essentielle pour la survie du parasite et a une activité de phosphatase pour l'acide phosphatidique, permettant la conversion de l'acide phosphatidique en diacylglycérol.

• L'inhibition de cette protéine induit des malformations membranaires ainsi que des défauts de cytokinèse et de réplication qui sont aggravés dans un environnement riche en lipide.

• Cette inhibition entraine aussi une biogenèse excessive de membrane nucléaire, endoplasmique et de complexes membranaires internes.

• TgLIPIN prévient la mort du parasite en le protégeant des effets toxiques des acides gras provenant d'une cellule hôte dans un environnement riche en lipide, en canalisant les flux d'acides gras issus de l'hôte et favorisant la synthèse de triglycérol pour la formation de gouttelettes lipidiques.

Ma contribution à ce travail inclut :

• Expérience de complémentation hétérologue prouvant l'activité phosphatase de la protéine

• Analyse des flux de lipide et lipidomique

• Illustrations et écriture de l'article.

SCIENTIFIC QUESTION OF THE THESIS

Hypothesis:

Apicomplexa parasites cause major human diseases such as malaria and toxoplasmosis, that still impose major economic and health burdens of large parts of the world. Large amounts of lipids are essential for parasite progeny and persistence within their hosts. FAs are central to lipid production and the parasites are unique in that they can scavenge FAs directly from the host cell and their environment and newlysynthesize FAs themselves via an exclusive prokaryotic type II fatty acid synthase or FASII within a non-photosynthetic essential relict plastid, named the apicoplast. The complex life cycles of these parasites require them to invade a multitude of different host under varied conditions, meaning they must be able to adapt to the host conditions to survive. Their prevalence and drug resistance emphasizes the need to understand the parasites biology and identify novel drug strategies. Recent work is only starting to uncover that these parasites can significantly adapt to the nutrient availability of the extracellular host environment (Zuzarte-Luís and Mota, 2018; [START_REF] Primo | The Extracellular Milieu of Toxoplasma ' s Lytic Cycle Drives Lab Adaptation , Primarily by Transcriptional Reprogramming[END_REF]Shunmugam et al., 2021;Shunmugam, Arnold, et al., 2022;Walsh et al., 2022). Many questions remain unanswered about the lipid metabolism of these parasites:

• How are FAs activated for their use in the lipid-processing pathways of the parasite?

• How does different nutrient environments affect the lipid synthesis within the parasite?

• How does the parasite recycle lipids and contribute to the dynamic remodelling of their lipid metabolism strategies?

My PhD project focused on answering the above questions by identifying three protein families, which we hypothesized play major roles in these mechanisms:

• Acyl-CoA synthetases (ACSs), which can activate FA for their metabolic use by the parasite

•

Phospholipases that can release FA from existing or recycled lipids

• Acyltransferases (ATs) that (re)incorporating FA to generate new lipid species

Aim and Objectives:

Their putative roles and importance in the lipid synthesis pathway are not fully understood as is critical to the propagation of the parasite.

I aimed to elucidate and characterize the roles of prioritized protein candidates in

Apicomplexan parasites, specifically Toxoplasma gondii and Plasmodium falciparum.

The completion of my PhD followed the objectives presented below:

1.

Generate molecular tools (CRISPR-Cas9) to characterize the lipid processing pathway in the two Apicomplexa models P. falciparum and T. gondii *Equal senior and corresponding authors. To whom correspondence should be cyrille.botte@univ-grenoble-alpes.fr ; yoshiki.botte-yamaryo@univ-grenoble-alpes.fr

ABSTRACT

Apicomplexa parasites cause immense health and economic burdens such as toxoplasmosis and malaria. They mostly comprise obligate intracellular 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 (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channelled towards parasite lipid storage and are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on an obligate metabolic activation mediated acyl-CoA synthases (ACS), which catalyse the thioesterification of FA to a CoA. Beside the essential functions of FA for parasite survival, the presence and roles of ACS is yet to be determined. Here, we identified that TgACS1 is a vesicular cytosolic protein (peroxisomal) protein which is involved in lipid metabolism of the parasite during low host nutrient conditions. The protein was present in cytosolic punctuates and revealed a host-nutrient-dependent role during both intracellular and extracellular tachyzoite life stages. We show initial evidence that this protein is involved in FFA metabolism for peroxisomal processes such as βoxidation.

IMPORTANCE

Toxoplasma gondii is an apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The as one of the key metabolic pathways for parasite survival and pathogenicity (1,2).

Toxoplasma gondii is capable of infecting virtually any nucleated cell from warmblooded animals, thereby reflecting its metabolic capacities and plasticity 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 (3,4), as signalling molecules participating in key events like active invasion and egress through timely microneme secretion (5-7) and as storage fuels, which plays critical role to maintain proper intracellular development (8,9). To cope with a continuous need for lipids within the different host cells environments, T. gondii has evolved so it harbours 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) (3,10), (ii) the ER-based FA elongation pathway ( 4) and

(iii) the eukaryotic type-I fatty acid synthase present in the parasite cytosol (10).

Additionally, the parasite is also heavily reliant on acquiring FA directly from their hosts (4,8,9,(11)(12)(13). Current consensus on parasite lipid metabolism suggests that both FA flux derived from apicoplast FASII (3,10), and FA obtained from direct scavenging from the host (4,8,9,(11)(12)(13), are critical for intracellular parasite viability. Interestingly the metabolic input of both FA sources can be regulated upon the host nutrient availability. Under high host lipid content, FASII can be downregulated and maintained at a basal level for the parasite to rely more on host scavenging. On the other hand, under low host lipid content, the FASII metabolic activity is significantly upregulated concomitantly with an upregulation of the scavenging of host membrane material, both to meet and maintain the parasite demand for lipids (13).

Interestingly, major phospholipid classes making the bulk of T. gondii lipid composition are obligate "patchwork molecules" composed of FA from the host as well as made de novo by the parasite (3). Most importantly, to optimise the use of host nutrient and avoid death by lipotoxic accumulation of lipids, the parasite channels the constant flux of scavenged FA towards its own lipid storages, i.e. lipid droplets (LD) in the form of neutral lipids, mainly triacylglycerols (TAG) (9). LD and their TAG content are then timely mobilized to provide an essential boost of FA for membrane biogenesis specifically during parasite division (9). Taken together, FA, and most importantly their acquisition, mobilization from sources/storages, and utilization are pivotal pathways for sustaining parasite survival.

One of the key biochemical steps required for general FA utilization and trafficking is their metabolic activation by thioesterification to a Co-Enzyme A (CoA). This metabolic activation step is catalysed by the essential acyl-CoA synthetase (ACS or acyl-CoA ligase) family (14). Biochemically, ACS catalyses a two-step reaction to form fatty acyl-CoA (Fig. 1a). The initial step involves the formation of an adenylated intermediate through the hydrolysis of an ATP molecule, thus releasing pyrophosphate (15). The ATP-activated enzyme then binds to the carboxyl group of an incoming 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 (15).

ACSs exist as different isoforms in eukaryotes depending upon on FA substrate specificity, (based on FA chain length and degree of unsaturation), metabolic or cellular function (FA storage, β-oxidation etc.). Humans have 26 enzymes that comprise the large ACS family activating short-chain, medium chain, long chain and very long chain fatty acid substrates (14). There are 9 long chain ACSs (LACS) that have been well characterized in Arabidopsis thaliana (16). 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 (14,17).

The phylum apicomplexa is actively engaged in FA metabolism and hence the role of the ACSs in parasite survival within its host cannot be undermined individually/apart from the host. 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 (10). C. parvum has been reported to have three AMP-binding domains containing long-chain fatty acid CoA ligases, of which CpACS1 and CpACS2 have been biochemically characterized as functional ACSs (18). Plasmodium falciparum on the other hand has a large family of ACSs encoded by 13 genes (PfACS1a-PfACS12), which functions remain to be determined (19). Interestingly, P. falciparum ACSs are also the only family of metabolic enzymes that are encoded by genes expanded into the sub-telomeric region, usually harbouring parasite virulence genes (19). Drugs specific to ACSs in C. parvum and P.

falciparum have shown potent parasite killing activity, thereby hinting that this enzymatic reaction is a potential Achilles heel for apicomplexans (18,20). Although the presence of an ACS was reported in T. gondii by bioinformatics analysis (21), the number of ACS or their functional roles in T. gondii are yet unknown.

Here, we identified that T. gondii encodes and harbour 7 putative ACSs by phylogenetic analysis. TgACS1 was shown to have a specific role for FA utilization when parasites face low nutrient environment, especially during extracellular stages.

Moreover, we found that TgACS1 belongs to the ACS bubble gum family and can only compensate a yeast homologue specific for beta oxidation. Therefore, this strongly suggests that TgACS1 is involved in providing energy through FA mobilization, potentially β-oxidation in a peroxisomal-like compartment in low nutrient content.

RESULTS

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

To identify putative members of ACS gene family in Toxoplasma gondii, we searched for genes bearing the typical AMP-binding domain found in eukaryotic ACS by mining the ToxoDB database (http://toxodb.org/toxo/). We were able to identify several candidate genes. Within these candidates bearing the AMP-binding domain, seven proteins were identified bearing highly conserved signature ACS motifs, named Motif I-V (15). We assigned 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. 1b). Phylogenetic analysis allowed the cladding of the different TgACSs to specific clusters based on their homology to other eukaryotic counterparts (Fig. 1b). TgACS1 cladded together with the human ACSs belonging to the bubblegum gene family (ACSBG), also known to be involved in β-oxidation, i.e. FA catabolic pathway allowing the generation of reducing power and energy to the cell (22).

TgACS1 has already been predicted to be involved in the possible βoxidation by its peroxisome transit sequence PTS (21). Based on their CRISPR-based phenotype scores (23) and predicted functions based on sequence homology and phylogenetic analyses, we decided to focus on a specific candidate TgACS1 with its putative role\as bubble-gum ACS, thus potentially involved in FA mobilization for a putative and longdebated β-oxidation in T. gondii.

The PTS sequence in TgACS is crucial for correct protein localization

In order to identify putative functional domains in TgACS1 protein sequence, Smart

Protein program (www.smart.embl-heidelberg.de) and Phyre 2 were used to compare to known domains. The presence of bubble-gum domain was identified in TgACS1 and this supports the phylogenetic analyses described earlier (Fig. 2a) suggesting similar functionality of this protein. We also identified a highly conserved peroxisomal targeting sequence (PTS) present in the C-terminal end of the protein (tripeptide, AKL). Other alveolates such as Hammondia and Neospora, and a known peroxisomal ACS in Saccharomyces cerevisiae also contain this specific PTS (Fig. 2b). Using Phyre 2 and a 3D crystal structure of a bubble-gum-domain-containing ACS found in Mycobacteria tuberculosis (24), we generated a 3D model prediction, which had a sequence identity of 51% with TgACS1 (Fig. 2c). These protein structures seem to have similarly placed hydrophobic regions suggested to be the AMP-binding cavity and similar placement of arginine residues which suggest common functionality (Fig. 2c ; (24)).

To characterize and identify the localization of TgACS1, we generated an AID-based (25) inducible knock down mutant of TgACS1 with HA-tag. Immunofluorescence analyses (IFA) revealed that this protein had a punctuated/vesicular cytosolic localization, which we originally thought to be overlapping with lipid droplets (LD) of the parasite (Fig. 2d). LTox staining, which stains LD, showed close association of TgACS1 with LD but no direct co-localization. A similar mutant line was developed lacking the predicted PTS1 (C-terminal AKL, consensus of the tripeptide is

[(S/A/C)(K/R/H)(L/M)]
). Immunofluorescence microscopy of both lines revealed that when the PTS signal is removed the protein loses its cytosolic/punctuated localization and instead localizes to the apical end of the parasite, close to the Golgi apparatus (Fig. 2e). Further detailed localisation analysis, however, shows no direct localization with the Golgi apparatus (GRASP45-RFP) or micronemes (anti-AMA1) of the parasite since it was thought to start aggregating in an anterograde manner towards the apical pole randomly (Fig. 2e).

TgACS1 is important for intracellular development in low nutrient environment

We also confirmed the protein size of HA-tagged TgACS1-iKD by Western blot analysis with its expected size of 100 kDa (Sup. 1b). The downregulation of TgACS1 by the addition of 100 µM auxin was also confirmed by Western blot and IFA analyses both after 30 min of treatment (1% FBS, Sup. 1b and Fig. 3a), with complete degradation of the protein within 1 h of treatment. To determine if the depletion of this protein influenced parasite growth, a replication assay was performed and showed a slight but not significant increase in small vacuoles (1-4 parasites per vacuoles) and decrease in larger vacuoles (10 parasites or more per vacuole) upon TgACS1 degradation (Sup. 1c).

Since the host nutrient environment affects the development and metabolism of the parasite as shown in our previous work (7,12), the parasites were grown in different concentration of FBS. Seeing that the phenotype of TgACS1 at normal FBS conditions (1%) was not indicative of essentiality, as proposed by the phenotypic score (33), we further investigated how the loss of this protein would affect the parasite in differential host nutrient environments. We thus performed plaque assays in the presence of different host nutritional conditions, i.e. at 0, 1, or 10% FBS in DMEM as previously described. A significant difference was seen in the growth of plaques under starvation lipid restrictive conditions (0% FBS) and in normal conditions (1% FBS, Fig. 3b).

Parasites grown in the presence of auxin in these two conditions showed to have less plaque area on average and hence a delayed growth in the absence of TgACS1. In 10% FBS, parasites were able to survive without the protein (Fig. 3b) suggesting that TgACS1 does not have an essential role in high nutrient conditions. Our results on parasite growth, confirmed that TgACS1 is involved in parasites development in a nutrient-dependent manner, this was further confirmed by relative quantification of mRNA levels of this protein (Sup. 1d), which showed an increase in expression of this protein in nutrient poor conditions and specifically in extracellular parasites.

Since TgACS1 grows differently in response to the nutrient availability, it is possible that its flux of storage may be altered. In addition, LDs are not only the main source for lipid storage, but they also can serve to peroxisomes mediated lipid-based energy conversion via β-oxidation (26). Hence, we decided to analyse how these lipid energy stores were affected under the suppression of this protein in different nutrient conditions. Here, instead of LTox, we used Nile red to stain the LDs in various nutrient content, (0-1% FBS). The results showed an increasing accumulation of lipid droplet number as FBS concentration increased but no difference in the number of lipid droplets per parasite with or without the TgACS1 protein (Fig. 3c). Closer analyses revealed that only in a high host nutrient environment was there no changes in lipid droplet volume (Fig. 3d), suggesting TgACS1 is not important in intracellular parasites in a high lipid content.

TgACS1 is important in FFA formation and TAG utilization in intracellular parasites depending on the availability of host nutrients.

Although there was an increase in the lipid droplet number by the change in the nutrient condition, there was no difference between the parasite with or without the TgACS1 protein.

Here, to determine if there is any change in the major lipid content and so as the lipid changes were reflected in the lipid metabolism of the parasite, we conducted lipidomic analyses by gas chromatography-coupled to mass spectrometry (GC-MS)

and separation of neutral lipid groups via high-performance thin layer chromatography (HPTLC) on TgACS1-iKD intracellular parasites with and without auxin.

Firstly, we examined the lipidomic profiles of intracellular parasites at 0-10% FBS (Fig. 3e) and noticed no changes in total lipid abundance when the protein was depleted, which was suspected since we saw little to no changes in LD formation in the nile red analyses (Fig. 3c-d). To further investigate the nutrient dependent role of TgACS1 we quantified the phospholipid and neutral lipid content of intracellular parasites in different nutritional conditions (namely 0-10% FBS, Fig. 3e-i, Sup. 2a-c, g/h).

The fatty acid profile of intracellular parasites in 0% FBS media revealed that under suppression of TgACS1 there was a significant increase in the mol% of C18:1 and decrease in the mol% of C18:0 (Fig. 3f). This suggests a role with-or higher substratebinding affinity to C18:0/C18:1. To further determine the precise function of TgACS1, the total lipid fractions were subjected to separation by HPTLC and the composition and content of total phospholipids (PLs), triacylglycerol (TAG, LD constituent), diacylglycerol (DAG) and free fatty acids (FFA) were quantified by GC-MS. This detailed lipidomic analyses revealed a slight but not significant increase in TAG levels and a significant decrease in FFA levels when the protein is degraded in 0% FBS (Fig. 3g). This suggests that FFAs are the final product of this enzyme, with a slight hint to TAG moieties being the substrate. At 1% FBS, intracellular parasites without TgACS1 displayed no changes in their total lipid fatty acyl profile. Additionally, there were no noticeable changes in neutral lipid content when the protein was knocked down under these same conditions (Fig. 3h-i). There were no changes in the fatty acid profile of the individual lipid groups in intracellular parasites when the protein was depleted in 1% FBS (Sup. 2b).

TgACS1 is important for extracellular parasite lipid droplet utilization

Over the general surveillance of TgACS1-ikD we noted that under normal 1% FBS conditions, IFAs indicated a punctuated cytosolic localization in extracellular parasites (Fig. 4a). mRNA quantification of this gene under different conditions indicated expression was highest in intracellular parasites but, equally as high in extracellular parasites at 0% FBS, or under low nutrient availability (Sup. 1d). This persuaded us to determine if we see the same trend in extracellular parasites and whether TgACS1 plays a role in lipid droplet utilization in these sub-optimal conditions. Interestingly, lipid droplet number/volume is higher in extracellular parasites as compared to intracellular parasites (2-3-fold), suggesting that the parasite increases lipid droplet formation in the absence of a host (Fig 3c,and Fig 4b). The number of lipid droplets per parasite at 0%

FBS and the lipid droplet volume at 1% FBS nutrient conditions were respectively increased in extracellular parasites under protein loss (Fig. 4b-c).

To determine if this observation is also present under complete loss of nutrients, extracellular parasites were incubated with PBS alone for less than 2 h (to avoid parasite death) and stained them with nile red (Sup. 1e). In PBS conditions, lipid droplet numbers were significantly less than in 1% FBS conditions, and further diminished when TgACS1 was degraded under auxin treatment in extracellular parasites. LD analyses on intracellular and extracellular parasites suggests a role of the protein in the utilization of lipid droplet constituents such as TAGs. It also suggests that this role is highly dependent on nutrient availability.

TgACS1 is important for extracellular parasite motility and energy production

Fatty acid stored in a form of TAG can be hydrolysed and used for energy production through the process of β-oxidation. We determined if TgACS1 could be involved in energy-production in the parasite. Therefore, we analysed the viability energyproducing state of the TgACS1-ikD parasites under 'stressful' conditions that may potentially induce β-oxidation i.e. without nutrients and outside the host. We performed parasite gliding (motility) assays of extracellular parasites in different nutrient conditions (0-1% FBS with and without glucose), wherein we measure the slime trail length formed by the release of SAG1. It revealed that under TgACS1 depletion, in all conditions, parasite motility in terms of average trail length (µM) was significantly decreased (Fig. 4d-e). When measuring the motility rate (nominal data) we notice that only at low nutrient conditions (0% FBS and PBS) is motility diminished for extracellular parasites (Sup. 1f). Interestingly it was noted that in the absence of glucose (which is the primary source for ATP-production, i.e., glycolysis) loss of the protein did not change the total lipid abundance of the parasites (Sup. 2j). This suggest that the role of TgACS1 is rather based on the level of FBS, hence nutrients and most likely its main constituent lipids, in the extracellular media.

This confirms the role of TgACS1 in energy-requiring processes like motility and emphasizes that the role is most important in low lipid conditions. The relationship between the parasite and the putative β-oxidation process occurring is uncharacterized and there is still much debate on whether the process even occurs in the parasite with or without a host. These data now suggests that the parasite is still able to produce energy to facilitate gliding in sub-optimal conditions, hinting that even without the host the parasite can produce its own energy from its own reserves.

Secondly, due the cellular phenotypic changes in terms of lipid droplet formation, we investigated the lipidomic profile of TgACS1 depleted extracellular parasites. By doing so we have also been able to first identify the lipidome of the intermediate extracellular phase of the T. gondii tachyzoite development. Surprisingly we noticed no changes in the total lipid abundance of extracellular parasites at 0-10% FBS (Fig. 4f) when

TgACS1 was depleted.

We noticed that the lipid content was higher (2-3-fold) in extracellular parasites than intracellular parasites (Fig. 2e and Fig. 3f), which reinforces the same observations seen in LD number (Fig. 3c-d and Fig. 4b-c). This suggest that more lipids are stored within extracellular parasites than when they exist within a host. Detailed investigation into the fatty acyl profiles of extracellular parasites in 0% and 1% FBS revealed changes in mol% of specific length FA. In no FBS conditions the loss of the protein results in a decrease of C14:0 (most derived from the apicoplast) and an increase in FA species normally scavenged from the extracellular environment/host (C18:1 and C20:1). A largely significant increase in C18:1 in 0% FBS again suggests a strong substrate specificity of the protein (Fig. 3g) as was seen for intracellular parasites (Fig. 3f). As in intracellular parasites, we again see a decrease in FFA levels in extracellular parasites in 0% FBS (Fig. 4h), affirming that FFA moieties may be the final product of the enzymatic reaction facilitated by TgACS1.

We also quantified the fatty acid abundance of extracellular parasites in different nutrient condition. In extracellular parasites at 1% FBS, under depletion of the TgACS1, there is a similar trend where in there is an increase in C18:0 and C16:0 and a decrease in C18:1, suggesting TgACS1 has a substrate specificity to these chains (Fig. 4i). Most interestingly, the detailed analyses of the neutral lipids again show a slight but not significant increase in TAG levels with a decrease in FFA moieties (Fig. 4j). These data collectively suggest TgACS1 plays an important role in low nutrient conditions, namely 0-1% FBS, specifically in the mobilization of acyl chains from TAG/lipid stores and furthermore the formation of FFAs.

Overall, these results suggest that TgACS1 plays a more important role in the activation of FFA in extracellular parasites under low nutrient conditions. Moreover, this protein seems to have a substrate specificity to longer, unsaturated FA species that are normally externally derived.

TgACS1 complements a yeast strain defective of peroxisomal acyl-CoA synthase function

In order to determine the acyl-CoA synthase activity of TgACS1, we performed heterologous complementation in yeast Saccharomyces cerevisiae strain defective of the acyl-CoA synthases. In yeast, it is known that acyl-CoAs can be made by the endogenous FAS pathway or by the activation of exogenous fatty acyl chains by ACSs. [START_REF] Johnson | Genetic Analysis of the Role of Saccharomyces cerevisiae Acyl-CoA Synthetase Genes in Regulating Protein N-Myristoylation*[END_REF] have shown that in yeast to major enzymes (Faa1 and Faa4) are involved in the activation of long chain acyl-CoA chains. Only when FAS is inactivated (with the addition of cerulenin) the double mutant strain can no longer grow even with exogenous FAs.

We transfected the mutant yeast strain (faa1∆faa4∆) with a pRS high copy (ura3) plasmid expressing TgACS1with a His tag. The WT and mutant strain transfected with the empty vector (no TgACS1) were used as controls. All experiments were performed in synthetic YNB media without uracil, supplemented with glucose (2%) and Tween20

(0.2%, to induce β-oxidation). Growth curves were determined at 30°C (Fig. 4). The WT strain displayed a phenotype as expected, where the inhibition of FA synthesis with the addition of cerulenin lead to a decreased growth rate of this strain. There was no rescued phenotype under cerulenin treatment and addition any FA species (Sup.

3), suggesting TgACS1 is not an ACS of this nature dependent on exogenously acquired FFAs (37).

This prompted us to investigate other types of ACS proteins found in yeast. ScPCS60

is an ACS involved in peroxisomal fatty acid metabolism of budding yeast. This protein is integrated in the peroxisomal β-oxidation system of this organism (38). Addition of doxorubicin (anti-cancer drug targeting peroxisomal β-oxidation) has shown to inhibit growth of yeast lacking this protein (39) on the ∆PCS60 yeast strain (Fig. 5). In the absence of doxorubicin, the ∆PCS60 strain grows optimally and similarly as expected with the empty vector plasmid and pRS-TgACS1-His. When treated with doxorubicin, the strain with the empty vector still grows put at a slower rate, but not to the same OD600 reading as the untreated cells. Growth is almost completely restored in the doxorubicin-treated ∆PCS60 strain complemented with pRS-TgACS1-His, suggesting that TgACS1 can rescue the effects caused by this drug. This strongly indicates that this protein has an acyl activating capacity and plays a pivotal role in peroxisomal βoxidation.

DISCUSSION

Acyl CoA synthetases catalyse a fundamental and limiting reaction in FA metabolism (Fig. 5a): 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 ( 14).

Here we identified 7 acyl-CoA synthetase (ACS) enzymes found in T. gondii parasites, all of which possess an AMP-binding domain (27,28). Multiple sequence alignment (Fig. 1b) of these proteins with known ACS proteins in other eukaryotic models suggest an overall identity of up to 30-40% specifically within the putative AMP-binding domains. Phylogenetic analysis of the identified TgACSs with known ACS from other eukaryotes clustered them to various clades, potentially based on their enzymatic substrate-specificity and function (Fig. 1b). 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 harbour 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 longchain (ACSL) and very long chain acyl-CoA synthetases (ACSVL) (15). These ACSL and ACSVL have the specificity to activate fatty acid up to 16-22 carbons in length (14). 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 (14).

Lastly, the bubble gum-ACSs called ACSBG gene family (also known as lipidosin) that can activate FAs of chain length 16-24 (29).

The diverse sub compartmental localization of putative Toxoplasma ACSs indicates non-redundancy 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 (30)(31)(32) and prostate ACSL1 localized at mitochondria, has a significant contribution to FA β-oxidation (28,33). ACSL3, which has been found on lipid droplets and ER, participates in FA uptake and glycerolipid biosynthesis (34). 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 (18).

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 voltagedependent anionic channel (VDAC) (33,35). No CPTa homologue exists in apicomplexa parasites (8), however recent data now provides evidence for the presence of an active acyl-CoA binding protein-2 (ACBP) that localizes to the mitochondria of the parasite (11,13) and TgACBP2 are p( 21)otentially 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. It is also possible that this parasite favours a peroxisomal β-oxidation system instead which can explain the loss of CPT1a but the existence of other β-oxidation proteins .

Amongst several functions of the ACSs includes channelling of FA towards energy yielding process of β-oxidation (14,16,36). The mouse ortholog of human HhsACSBG1, 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 (22). Interestingly, based on phylogenetic analysis

TgACS1 clusters together with the hsACSBG-gene family known to be involved in βoxidation (14). 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 (37,38). Proteins Our complementation experiments of an acyl-CoA synthase deficient yeast strain (Fig. 5), illustrates acyl-CoA synthase activity of TgACS1, but that that this protein has a preference in the activation of FAs (namely C18:1 which induces expression of ScPCS60) destined for β-oxidation, as all experiments were preformed using Tween20

(a polysorbate with a C12:0, lauric acid attached), which induces β-oxidation. These experiments strongly indicate that this parasite may have a very 'minimalistic' version of β-oxidation process in the parasite peroxisome, but it would be critical to investigate these metabolites under conditions that induce the process such as nutrient deprivation. Co-localization studies with peroxisomal markers like catalase and lipidomics can provide further confirmation.

In this study we show the first lipidome of extracellular parasites and parasites with differing host nutrient conditions. These data now show that extracellular parasites seem to have 2-3-fold more lipids internalized when extracellular as opposed to being within a host (Fig. 3e and Fig. 4f) and are supported by the increase in lipid droplet numbers of extracellular parasites (Fig. 3c and Fig. 4b). The consistent increase in TAG and decrease in FFA levels under the depletion of TgACS1 in intracellular and extracellular parasites, specifically in low nutrient conditions (0-1% FBS), highlights the nutrient dependent role of this protein in the mobilization of TAG moieties into activated FFAs. FFAs can be hydrolysed from TAG molecules via phospholipases. Thereafter we suspect ACSs are responsible for the activation of these recycled FFAs for other metabolic process. If these FFA are not incorporated, they will become toxic to the parasite cell. Hence why we see an accumulation of LDs as an attempt by the parasite to escape lipotoxic death.

The substrate-specificity of this protein to long chain fatty acids such as C18:1 seen in the lipidomic analyses of mutant parasites was reinforced by the yeast complementation experiment (we supplemented with C18:1 FFA), which showed growth of the ∆TgACS1 complemented mutant only with this FFA. The decrease in motility under low nutritious conditions additionally supports TgACS1 being involved in the utilization/mobilization of TAGs for energy production in non-ideal conditions (Fig. 5b). Recent findings on a putative ACSBG protein like TgACS1 in the microalgae Microchloropsis gaditana, shows a similar increase in TAG levels when the protein is disrupted and hypothesizes a role in TAG utilization and β-oxidation (37). It has been reported that one of the sources of energy for the T. gondii motility is gamma aminobutylic acid (GABA, ( 40)). In our study, there was always some glutamine included which could be used to serve for the TCA cycle directly or via GABA synthesis. In comparison to the ultimate nutrient deficient condition, i.e. PBS, the parasite probably still had very low motility DMEM medium without glucose or FBS. Here, the presence of TgACS1 with FBS supplying to the possible beta oxidation showed the high increase of motility suggesting that the TgACS1 dependent activation for the beta oxidation is the major supply for the energy to the parasite motility.

Further experiments in the role of TgACS1 in peroxisomal β-oxidation are necessary in understanding its role in lipid metabolism. We already know from previous work that the nutritional availability (9,13)and governs the parasites survival. This work brings insight to the parasites ability to adapt to changing host environments and in depth understanding of the diverse roles of TgACSs in this process to allow for parasite propagation. 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), caninum (NCLIV_018500). First step involved the curation of these protein sequences.

MATERIALS AND METHODS

Protein sequence analysis and

P. falciparum_PfACS12 (PF3D7_0619500), A. thaliana_LACS1 (At2g47240), A. thaliana_LACS2 (At1g49430), A. thaliana_LACS3 (At1g57920), A. thaliana_LACS4 (At4g23850), A. thaliana_LACS5 (At4g11030), A. thaliana_LACS6 (At3g05970), A. thaliana_LACS7 (At5g27600), A. thaliana_LACS8 (At2g04350), A. thaliana_LACS9 (
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 Tir as well as the mutant strain were cultured 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 inducible knockdown line for TgACS1

A C-terminally tagged HA line with inducible post-translational protein degradation was generated using the Auxin-inducible Degron system (AID). A 2685 bp homology region of the C-terminal end of TgACS1 excluding the stop codon was amplified using the primers in Table 1. A guide sequence (Table 1) was selected near the C-terminal end and was less than 100 bp away from at least one homology region. The guide sequence was inserted into Cas9 U6 universal plasmid Q5 mutagenesis.

The PCR product and the Cas-9 guide plasmid were used for transfection of a RH TIR1-3FLAG parental line (34). After 24 hours the newly transfected line was treated with 25 μg/ml mycophenolic acid and 50 μg/ml xanthine. Parasites were selected with the drug and cloned by limiting dilution. Screening of parasite clones for correct insertion of the mAID sequence, HX drug cassette and overall insertion/orientation were done with the primers in Table 1. All PCRs were performed with TaKara primestar Max polymerase. The knockdown of TgACS-1 was induced with a final concentration of 100 µM of indole-3-acetic acid (auxin) made up in 100% ethanol.

Table 1: All PCRs were performed with TaKara Primestar Max polymerase.

Immunofluorescence assay

Primary antibodies anti-HA (Rat, Roche), anti-IMC1, anti-SAG1 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. The fluorescence was visualized using fluorescence microscope (Axio Imager 2_apotome; ZEISS).

Nile red staining of lipid droplets

Parasites were allowed to infect and grow in confluent monolayer HFF grown on coverslips, in the +/-ATc or Auxin conditions for 24 h in 0%/1%/10% FBS ED1 medium.

As with IFA, these coverslips were fixed using 2.5% PFA, permeabilized with 0.25% triton X-100. Thereafter, coverslips were incubated for at least 1 h with nile red (or LipidTOX™ HCS, 1 : 200) 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) was used on the Axio Imager. Quantification of lipid droplet number and volume was preformed based on iterative image processing as described by Exner et al., 2019 (41).

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) (Van Dooren et al. 2008) 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 TgACS1-iKD and TgAC2-iKD were grown for plaque assay in the presence or absence of ATc or auxin (0.5 μg ml -1 or 100 µM) 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 phosphatebuffered saline (PBS) wash.

Replication assay-The parasites were grown for two days with or without ATc or auxin, 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 toto remove any uninvaded parasites and promote synchronized replication. ATc or auxin (same concentration as above) was added at the outset of the experiment, allowing the treatment for 24 h, alongside control parasites without auxin. These coverslips were then fixed and processed for IFA wherein the parasite number per parasitophorous vacuole was analysed.

Motility assay-Slides were prepared before start of the experiment whereby, 1.5 mm glass slides were flamed and smeared with 0.1% polyethyleamine. Slide were placed in 6 well plates and incubated with a layer of FBS for 2-3 hours at 37⁰C. Freshly egressed TgACS-1 iKD parasites were passes through a 3 µM filter and spun down for 10 mins at 1800 rpm. Cells were washed with PBS three times and resuspended in 1 ml of ED1 media (100 mM HEPES) or PBS (100 mM HEPES), in conditions with or without 100 mM auxin. Before placing the cell suspension, slides were washed three times with PBS to remove excess FBS on the glass slides. Cells where incubated added to the wells for 10 mins at room temperature to allow cells to settle and for another 1 hour at 37⁰C. Samples were fixed with 5% paraformaldehyde/PBS for 15 min and processed for an IFA using anti-SAG1 antibody. The experiment was performed three independent times.

Relative mRNA expression quantification-RH TIR1-3FLAG parasites were grown on confluent monolayer of HFF in flasks (175 cm 2 ) and allowed to grow for 48 hours (mainly intracellular) or until completely egressed (extracellular parasite) in 0, 1, or 10% FBS normal DMEM conditions. Parasites were then harvested by scaping cells of the bottom of the flask and moved to 50 ml tubes; intracellular tachyzoites were pushed through a 0.2 mm needle and syringe filtration with 3-μm pore size membrane to remove host cell debris. A NucleoSpin RNA kit (Macherey-Nagle) was used for mRNA extraction and whereafter cDNA was synthesized using a SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR kit. Primers specific (Table 1) to each gene of interest, including a house keeping gene GAPDH1 (TGGT1_289690) which was used to calculate the relative mRNA expression in the different conditions used in this study.

Lipidomic analysis

The parasites were grown for 24 h or until completely extracellular in conditions of +/auxin on the confluent monolayer of HFF in flasks (175 cm 2 ). For extracellular parasite lipid analyses completely, egressed parasites were collected, and host cells were filtered out with a with 3-μm pore size membrane, after which parasites were incubated for 6 hours in the respective media conditions 0/1/10% FBS). At each time point, parasites were also 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 analysed by GC-MS as previously described [START_REF] Dubois | Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages[END_REF]. 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 one-dimensional 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.
Total PL, 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 analysed by GC-MS (Agilent).

Yeast complementation assay and growth curves

A pRS426-MET25 vector was used as the backbone for a plasmid containing the entire cDNA sequence of TgACS1. The plasmid was generated by GeneCust by cloning the sequence of interest using EcoRI and KpnI restriction sites. All strains were transfected either with this plasmid of the pRS426-MET25 vector lacking TgACS1 as a control.

The isogenic S. cerevisiae strains WT (YB332: MATa ura3 leu2 his3A200 ade2 lys2-801) and faa1∆faa4∆ (YB525: MATa ura3 leu2 his3D200 ade2 lys2-801 faa1∆::HIS3; faa4∆::LYS2) used in this study were kindly provided by Dr. Juliette Jouhet (CEA, Grenoble, France) and have been described earlier (52,53). After transfection, all the strains described in the present work were grown on synthetic media containing 6. Since no complementation was seen in the aforementioned strains, we began to investigate another acyl-CoA synthetase activities. We transfected the pRS426-MET25-TgACS1 plasmid into a yeast strain lacking a peroxisomal-CoA synthetase, ∆PCS60 (38,39) These experiments were also performed in TWEEN 20, with and without 6 µg/ml doxorubicin; a cancer-drug inducing lipid peroxidation and peroxisome proliferation in yeast cells (23,54). OD600 were calculated as described above.

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 FBS and not 1% FBS when the protein is degraded (auxin treatment, red). c) Further nile red analyses with (grey) and without (red) the protein shows that LD volume is increased at 1% FBS conditions for extracellular parasites. d) Motility assay using anti-SAG-1 revealed that (right) that at 0-1% FBS with glucose and e) without glucose conditions the length of slime trails per extracellular parasite is significantly diminished under suppression of TgACS1. f) Total lipid abundance of extracellular parasite in 0/1/10% FBS. g) Fatty acid (FA) profile (mol%) of extracellular parasite in 0% FBS with (grey) and without (red) TgACS1. h) Neutral lipid mol% of total lipid abundance in extracellular parasites in 0% FBS without (grey) and with (grey) auxin treatment, namely triacylglycerol (TAG), free fatty acid (FFA), diacylglycerol (DAG) and phospholipid (PL). i) Fatty acid (FA) profile (mol%) of extracellular parasite in 1% FBS with (grey) and without (red) TgACS1. j) Relative abundance ratios calculated for TAG, FFA, DAG and PL levels without/with the protein in 1% FBS in extracellular parasite.

Error bars indicate standard deviation (SD), Experiments were conducted in triplicates, unpaired t-test p values are only indicated when statistically significant. In the absence of doxorubicin, the ∆PCS60 strain grows (30⁰C for 32 h) as expected with the empty vector (red) and pRS-TgACS1-His (black). When treated with doxorubicin, the strain with the empty vector (blue) grows put at a slower rate. Growth is almost completely restored in the doxorubicin-treated ∆PCS60 strain complemented with pRS-TgACS1-His (green), suggesting that TgACS1 can rescue the effects of doxorubicin. • Ecriture de l'article et création des illustrations.

regulates replication and growth of Toxoplasma tachyzoites. Lipidomic analysis of parasites lacking TgACS3, suggests its role in activation of host-derived FAs directed towards parasite phospholipid and triacylglycerol synthesis in high lipid conditions.

Altogether, the results presented here confirm the importance of ACS enzymes in T.

gondii parasite survival and lipid activation and metabolism.

INTRODUCTION

Apicomplexa parasites are a group of unicellular eukaryotes that include obligate intracellular parasites responsible for major human and cattle diseases. These include Toxoplasma gondii, Plasmodium sp. and Cryptosporidium parvum, which are causing agents of the infectious diseases toxoplasmosis, malaria and cryptosporidiosis, respectively. There is currently no fully efficient vaccines, and, depending on the parasite and the related diseases, current treatments can be toxic, losing efficiency and or emergence and fast spreading of resistance amongst parasite populations.

There is thus an urgent need to renew our therapeutic arsenal to further push for the eradication of these important diseases. One important step for this is to understand and identify the key metabolic pathways sustaining host-parasite interactions and parasite survival. Metabolic pathways allowing nutrient acquisition, intracellular development, and parasite propagation represent ideal targets for drug development.

Lipid synthesis, acquisition and homeostasis have been shown as one of the key metabolic pathways for parasite survival and pathogenicity (1,2).

T. gondii is capable of infecting virtually any nucleated cell in mammals and birds, thereby reflecting its metabolic capacities and plasticity 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 (3,4), as signaling molecules participating in key events like active invasion and egress through microneme secretion (5,6) and as storage fuels, which plays critical role to maintain proper intracellular development (7,8). To cope with a continuous need for lipids within 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) (4,9), (ii) the ER-based FA elongation pathway ( 10) and (iii) the eukaryotic type-I fatty acid synthase present in the cytosol (9). Additionally, the parasite is also heavily reliant on acquiring FA directly from their hosts (7,8,(11)(12)(13)(14). Ongoing research on parasite lipid metabolism suggests that the FA flux derived from both apicoplast FASII (4,9) as well as from direct scavenging from the host (10,(13)(14)(15) falciparum (16). On the other hand, lipid scavenging is a constant phenomenon that renders FASII dispensable when the nutrient/lipid content is in excess in the host.

Furthermore, this constant flux of FA scavenged from the host, which can be lipotoxic for the parasite, needs to be used timely for phospholipid synthesis and membrane biogenesis, specifically during parasite division (8,19,20)The parasite accomplishes this by directing excessive scavenged FA towards triacylglycerol (TAG) formation which facilitates the biogenesis of lipid droplets (LD). These LD can be present throughout the asexual intracellular developmental stages and can be mobilized specifically during parasite division.

FAs need to be metabolically activated to be used by the cell for trafficking, metabolic utilization, or any other cellular function. This key biochemical step is performed by thioesterification of the FA to a Co-Enzyme A (CoA) that is catalyzed by the essential acyl-CoA synthetase (ACS or acyl-CoA ligase) family (21). Biochemically, ACSs catalyze a two-step reaction to form fatty acyl-CoA (Fig. 1a). The initial step involves the formation of an adenylated intermediate through the hydrolysis of an ATP molecule, thus releasing pyrophosphate (22). The ATP-activated enzyme then binds to the carboxyl group of an incoming 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 (22).

The phylum apicomplexa is actively engaged in FA metabolism, and hence a putatively critical role of ACSs for parasite survival is highly expected. Any putative ACSs candidate encoded in the Apicomplexa genome can theoretically activate FAs derived from both de novo synthetic machinery and/or from the host. Such importance and expected functions are supported with the existence of ACS encoding genes in the genome of Cryptosporidium and Theileria, which lack the de novo fatty acid biosynthetic machinery (9). Cryptosporidium parvum has been reported to have three AMP-binding domain containing long-chain fatty acid CoA ligases out of which two CpACS1 and CpACS2 have been biochemically characterized as functional ACSs (23). Plasmodium falciparum on the other hand has a large family of ACSs encoded by 13 genes (PfACS1a-PfACS12) (24). They 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 (24). 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 (23). Nothing is currently known on the function of ACSs in T. gondii.

We have identified 7 enzymes in Toxoplasma gondii, which we classify as putative acyl-CoA synthases that have non-overlapping localizations, suggesting non redundant roles (TgACS( 25)). In this study, we identified one of them, TgACS1, whose phenotypic score initially suggested it as not important for parasite growth. However, we further showed that the protein's function becomes critical under specific physiological conditions in the host. We reveal that it actually activates FA for their mobilization and use by a peroxisomal-like pathway to provide energy to the parasite under poor/deleterious host nutritional environment, notably during the tachyzoites extracellular stages. This indicates that the role of such enzymes might be directly linked to the capacity of the parasite to have a strong metabolic plasticity upon host nutritional conditions that strongly governs the metabolic pathways chosen by the parasite (1,2,8,17,(25)(26)(27)(28). The function and potential nutrient-dependent roles of the other ACSs in T. gondii are still unknown. Here, we reveal the essential function of another ACS candidate, TgACS3 (29).

Downregulation of

RESULTS

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

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 (30).

Immunofluorescence assays (IFA) using anti-HA antibody revealed that the 7 TgACSs localize to distinct sub-cellular compartments of the parasite (Fig. 1a). TgACS1, TgACS6 and TgACS7 were localized widely within the parasite cytoplasm, mostly as vesicular/punctate patterns. TgACS2 localized in a compartment reminiscent of the parasite mitochondria. TgACS3 was broadly localized at the cytosol, accumulating around, and avoiding the nuclear area of the parasite. TgACS4 showed a distinct and interesting peri-nuclear/ER-like localization. Finally, TgACS5 displayed a cytoplasmic localization at, particularly enriched at the basal end of the parasite. Such specific and diverse localizations suggest non-redundant functions of ACSs within the different endomembrane compartments and cytoplasm of T. gondii similarly to the ACSs in other eukaryotes, which have different fatty acid substrate specificities and functions depending on their differential localizations (21,31,32). The endogenously tagged parasite lines were further confirmed by western blot analysis using anti-HA antibody (Sup. 1b). The migration of all the TgACS-HA was in accordance with their annotated protein sizes on ToxoDB, except for TgACS2, which migrated approximately 10 kDa less than predicted.

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

To determine the function and importance of TgACS3 (TGGT1_310080), which is predicted to be important for intracellular growth with a phenotypic score of -2.53 (29,33), we generated an inducible knockdown line of TgACS3 (Fig. 2) by promoter replacement using the Tet-off system in TATi_ΔKu80 background (34,35) with an additional N-terminal HA-tagging. Western blot and IFA analyses both confirmed the downregulation of TgACS3-iKD-HA (Fig. 2a,b) parasite line by anhydrotetracycline (ATc) treatment. Complete protein depletion was observed at 48 h after ATc treatment for TgACS3 (Fig. 2a).

IFA confirmed TgACS3 is located at the cytosol/perinuclear compartment (Fig. 2b).

The disruption of TgACS3 had a direct deleterious effect on tachyzoite intracellular morphology, especially on large vacuoles (>10 parasites) where parasites appeared round and unhealthy (Fig. 2b, lower panel: phase). The growth phenotype was confirmed by a replication assay where TgACS3 depleted parasites had significantly fewer large vacuoles (7-10 parasites) and a significantly higher number of smaller vacuoles (3-6 parasites) (Fig. 2c), indicating that intracellular propagation was significantly impaired in the mutant. 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 could sense host nutritional status and grow better with higher levels of host nutrient (10% FBS) (14).

The disruption of TgACS3 strongly disrupted 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 the growth phenotype was significantly lesser than the control (Fig.

2d, e). 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. 2d,e).

Furthermore, TgACS3-iKD parasites also displayed a significant, yet mild egress defect in the presence of ATc, which may or may not be an underlying effect of replication defect (Sup. Fig. 1b).

To further determine the cellular phenotype induced by the absence of TgACS3, we monitored the content of parasite lipid storage, i.e. lipid droplets (LD), which play an important role for channeling the massive flux of host scavenged FA. Nile red staining of TgACS3 intracellular parasites at 0%, 1% and 10% FBS indicated that the disruption of TgACS3 induced a significant reduction in the number of lipid droplets per parasite (Fig. 2f,g). This suggests that TgACS3 plays a role in the formation of neutral lipids and storage either directly or indirectly.

Disruption of TgACS3 leads to decrease of TAG levels in a high host nutrient environment

Our data 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 GCMS approaches. We first determined the overall 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 FA abundance of TgACS3-iKD (+ATc and -ATc) (Fig. 3a). However, the detailed profile of the total FA composition showed a significant increase in palmitic acid (C16:0) with a decrease in C20:1 content in the mutant (TgACS3-iKD +ATc) (Fig. 3b). To assess function of TgACS3, the total lipid content was separated by HPTLC, and the composition and content of phospholipids (PL), free fatty acid (FFA), and storage lipid triacylglycerol (TAG) were quantified by GCMS. Interestingly, this detailed lipidomic analysis FFA were significantly increased in the mutant (TgACS3-iKD +ATc, 1% FBS) (Fig. 3c). The FFA excess in TgACS3-iKD mutant (+ATc) correlated with the growth defect phenotype in the presence of 1% FBS (Fig. 2e). However, disruption of TgACS3 resulted in no changes in TAG levels (Fig. 3c). Quantification of the FA composition of the FFA fraction revealed significant decrease in short chain FAs like C14:0 and C16:0 and an increase in long chain FAs like stearic acid (C18:0) and C22:1 under the depletion of TgACS3 (Fig. 3d). Analysis of the FA composition from TAG and DAG levels in the TgACS3-iKD mutant (+ATc), showed no significant changes in individual FA molecular species (Sup. Fig. 3a,b). Overall, this suggested TgACS3 depletion within the parasite, leads to an increase in inactive FFA, likely due to the loss of ACS activity to incorporate FA, potentially leading to a lipotoxic effect causing parasite death.

The exaggerated growth defect seen at 10% FBS under the suppression of TgACS3 (Fig. 2d, e) lead us to further investigate the lipidomic profile of parasites lacking this protein under a high lipid content. The total lipid FA composition analyses revealed that there was an increase in FA chains that are normally scavenged directly from the host (C18:1, C20:1, C20:4, Fig. 3e) (8,14). Investigation of neutral lipids and PL levels by 1D-HPTLC, revealed a significant decrease in the relative abundance of TAG levels without and with TgACS3 (Fig. 3f). There was an increase in C12:0 and decrease in C16:0 FA chains in the TAG levels when the protein was suppressed (Fig. 3g), suggesting that most of the TAGs are coming directly from the parasite and not scavenged as lipid droplets from the host ( 8). An increase in the relative abundance of C18:0 and decrease of C18:1 in DAG levels was observed (Fig. 3h). Despite no changes in DAG and PL relative abundance in the absence of TgACS3, there was a general trend of a decrease in unsaturated FAs chains (C18:1, C20:1) and a compensating increase of saturated FAs (C18:0, C16:0) in both lipid fractions (Fig. 3h,i).

TgACS3 is responsible for channeling the bulk of host scavenged FA

Based on the lipid data showing major effect of TgACS3 depletion on long chain fatty acids (>16:0/18:0), previously shown to be mostly scavenged from the host (8,14) These results prompted us to further examine the source of FA dealt with/activated by TgACS3 within the parasites. We performed novel flux analysis approach, using U- 13 C-glucose (Glc) combined to GCMS-lipidomics to monitor and quantify the FA scavenged from the host, i.e. the parasite "FA scavengeome" (8). 13 C-Glc-pre-labelled host cells were infected with TgACS3-ikD parasites (+/ATc) in the presence of normal culture medium, which contains regular 12 C-Glc (8). The total lipid content was extracted from the parasites to determine the amount of 13 C incorporation to each FA originating from the host. The wild type TgACS3-ikD parasites were able to scavenge fatty acids from the host, with a significant decrease in host-derived C18:1 in the absence of the protein (Fig. 4a). There was a decrease in scavenging of C16:0 and increase in C16:0 and C18:1 of TAG and FFA pools, respectively (Fig. 4b,c). There was an overall decrease in FAs scavenged from the host for DAG (C16:0) and PL (C16:0, C18:1) lipid pools (Fig. 4d,e) in 10% FBS. This data suggests that the TgACS3 majorly acts on FA scavenged directly from the host especially in a high host lipid environment balancing between PL synthesis and lipid storage. The increase in host derived FFA suggests without this protein they cannot be incorporated to form PL and other storage lipids like DAG/TAG.

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 (21). We prioritized TgACS3, due to their largely distinct roles in lipid metabolism and nutrient adaptation in other models (nutrient scarcity and excess) and its high predicted phenotypic score (25,29,(36)(37)(38)(39).

Eukaryotic TAG biosynthesis occurs as the last step of the glycerol-3-phosphate pathway by the sn-3 acylation of DAG by a diacylglycerol-acyltransferase (DGAT, Fig. 5a) (40,41). Here we tested the roles of TgACS3 this process by comparing the levels of neutral lipids within the reservoir organelles called lipid droplets by Nile red staining (42). This suggests that TgACS3 plays a role in the formation of neutral lipids and storage either directly or indirectly.

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 (40). Like 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. 3c). We see a drop in TAG levels under depletion of the protein in 10% FBS which is supported by the droplet in lipid droplet formation by nile red staining (Fig. 2g and 3g). Lipidomic analysis of TgACS3-iKD +ATc illustrated an upregulation of the normally FASII-derived fatty acids to compensate for the decrease in host-derived fatty acids like C20:1. In a high host nutrient environment we see increases in the total lipid abundance of even more long chain unsaturated FAs that are normally host-derived like C18:1, C20:1 and C20:4. This suggests that TgACS3 probably is a long chain fatty acyl-CoA ligase which thioesterifies long chain FA scavenged from the host rather than the apicoplast-FASII specific short chain fatty acids (C14:0 and C16:0). This was supported by our 13 C-Glc based flux analyses, where we were able to identify whether the increase of FFAs is host-derived. There was an overall decrease in host-derived FAs for total lipids, which accounted for the decrease in host-derived FAs of TAG, DAG and PLs pools (Fig. 4).

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) and more specifically an increase in host-derived FFAs (Fig. 5). 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, and instead of an equilibrium in lipid storage and synthesis we see a decrease in PL synthesis and lipid storage. 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. This led to 'over scavenging' of host FFAs, which seemed to accumulate instead of being used to form PL/DAG or TAGs in the absence of this protein (Fig. 5).

Despite having a negative phenotype score on Toxoplasma genome wide CRISPR-Cas9 screen( 29), 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 TgACSs when TgACS3 is depleted. However, the replication assay and egress defect demonstrated the importance of TgACS3 for the parasite intracellular development.

Inflammatory cytokine (TNF-α) elevation in humans, seems to induce the expression of ACSL1, ACSL3, and ACSL5 (44). Increase ACSL3 levels were also accompanied with increase oleoyl-CoA (activated FA C18:0) levels, mirroring our results of increased oleic acid (FFA) when TgACS3 is absent. The downstream effect of TNF-α increasing ACSL3 levels promoted lipid droplet formation in human endothelial cells which may also be the case in TgACS3 favouring activation of FFA to be used for TAG formation (44). In a recent study, ACSS3 (the short chain paralogue of ACSL3) was identified as a gene regulating the stability of lipid droplet-associated protein, PLIN3, and causing the reduction of LD deposits in the prostate (45). These studies suggest that ACS3

proteins are critical of LD metabolism and interact with partner proteins to regulate FFA activation and TAG formation.

This work brings insight into the nutrient-dependent role of TgACS3, as it is different from TgACS1 and most probably the other ACSs that exist in T. gondii parasites (25).

This protein is important in a high nutrient environment, again illustrating the parasite adapts different metabolic pathways in different conditions, rending them highly plastic (1,2,8,(26)(27)(28).

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 the mutant strains were cultured 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 an inducible knockdown line for TgACS3

For generation of an inducible knockdown parasite line for the gene TgACS3, plasmid pPR2-DHFR ( 46) was used. For homologous recombination in RH-ΔKu80 TATi strain two separate homology flanks/regions were chosen. The 5' flank was amplified 1298 bp upstream of the TgACS3 start codon using the primers in Table 1. The PCR product was ligated 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 in Table 1. 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.

The resulting vector were linearized with 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 in Table 1 in the combinations described in supplementary figures (Sup. 4f). All PCRs were performed with TaKara Primestar Max polymerase. The knockdown of TgACS3 was induced with 0.5 μg ml -1 of anhydrotetracycline (ATc).

Immunofluorescence assay

Primary antibodies anti-HA (Rat, Roche), anti-IMC1, anti-SAG1 were used at dilutions 1:500, 1:1000 and 1:1000 respectively. Secondary Alexa Fluor 488-and 546conjugated 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).

Nile red staining of lipid droplets

Parasites were allowed to infect and grow in confluent monolayer HFF grown on coverslips, in the +/-ATc or Auxin conditions for 24 h in 0%/1%/10% FBS ED1 medium.

As with IFA, these coverslips were fixed using 2.5% PFA, permeabilized with 0.25% triton X-100. Thereafter, 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) was used on the Axio Imager.

Quantification in +/-ATc or Auxin conditions were done by counting the no. of lipid droplets per parasite.

Western blot analysis

Parasites were harvested for western blot after complete egress from their host. 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) ( 47) 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 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-Similar to 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 processed for an IFA using anti-SAG1

antibody.

Lipidomic analysis

The parasites were grown for 48 h or until completely extracellular in conditions of +/-Auxin/ATc on the confluent monolayer of HFF in flasks (175 cm 2 ). For extracellular parasite lipid analyses completely, egressed parasites were collected, and host cells were filtered out with a with 3-μm pore size membrane, after which parasites were incubated for 6 hours in the respective media conditions 0/1/10% FBS). Parasites were also 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 [START_REF] Dubois | Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages[END_REF]. 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 one-dimensional silica gel high-performance thin layer chromatography (HPTLC, Merck). Total PL, 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).

Tracking host-derived fatty acids -(monitoring parasite scavenging capacities)

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 (10% FBS). These 13 C-pre labelled HFF were then infected with

TgACS3-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 analysed initially for HFF to check labelling of the metabolites (8). Later, the same was calculated for parasites to confirm direct uptake of 13 C-labelled fatty acids from the host.

Statistical analysis for all experiments

Entire graphical data for this study was generated using GraphPad prism software (version 8). 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=***.

Table 1

TgACS Gene ID Primers (gene specific sequence Les résultats principaux sont les suivants :

• PfPNPLA2 est localisée dans la mitochondrie ainsi que dans l'apicoplaste du parasite.

• Cette protéine est importante lors de la phase de reproduction asexuée et spécifiquement dans des conditions où les lipides sont limitants.

• Des analyses lipidomiques par GC-MS indiquent que la déplétion de cette protéine affecte seulement les profils lipidomiques des parasites au stade annulaire ou schizonte. This parasite undergoes a complex lifestyle in human red blood cells, hepatocytes, and mosquitoes. This renders this organism metabolically plastic, allowing the parasite to survive in very different host environments. One specific nutrient that is consistently needed, especially when the parasite divides to form the new progeny (hence allowing for disease propagation), is lipids. Parasite lipid metabolism relies on scavenging host resources and parasite lipid synthesis/recycling upon physiological variation of available nutrients.

Therefore, enzymes manipulating lipids constitute pertinent drug targets. Here, we show that one such lipase allows the parasite to recycle existing lipids for survival when host resources are lacking. This brings novel insight into how the parasite can sense its host environment to metabolically adapt, hence revealing potential hidden drug target. This will ultimately contribute to the fight against malaria.

ABSTRACT

Plasmodium falciparum is an Apicomplexa parasite causing human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Current evidence shows that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly formed parasites. Parasite membrane homeostasis is achieved by an essential combination of de novo parasite lipid synthesis/recycling and host lipid scavenging. Recent data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possess an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase

PfPNPLA2, for which we generated an GlmS-inducible knockdown strain and investigated its role during blood stages. Interestingly, the mitochondrial PfPNPLA2 had a significant parasite replication and survival defect under low host lipid content. Lipidomic analyses revealed that PfPNPLA2 specifically degrades parasite phosphatidylglycerol to generate lysobisphosphatidic acid.

PfPNPLA2 knockdown resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite PG to sustain optimal asexual development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

INTRODUCTION

There are 241 million cases of malaria per year worldwide, of which 627 000 (World malaria report, 2021) result in death, mostly children in Sub-Saharan Africa and South-East Asia (1). Malaria control has struggled to improve due to the rapid emergence of drug-resistant strains, and even more considering the current COVID19 pandemic, which dramatically hampered the current situation.

The global and rapid emergence of drug resistant parasite strains has made the search for novel drug targets even more urgent and pertinent in recent years (2). Malaria is caused by an obligate intracellular parasite called Plasmodium spp, which belong to the phylum Apicomplexa. The clinical symptoms and lethal consequences of the disease are due to the asexual intracellular development of the parasite within the host red blood cell. These parasites must develop and divide within erythrocytes and understanding the metabolic interactions by which the parasite can acquire essential host nutrients, especially during blood stages, is crucial to identify key pathways that could be targeted as potential therapies.

Transmission of malaria parasites is achieved by a blood feed of an infected female Anopheles mosquito on a human host, releasing Plasmodium spp.

sporozoites that travel towards the liver where they invade hepatocytes.

Parasites then undergo an intense asymptomatic asexual schizogonic division cycle, generating tens of thousands of parasites within hours. Newly formed merozoites then egress hepatocytes to enter the blood flow where they can invade host red blood cells to embark on the asexual intraerythrocytic stages to proliferate. During the intraerythrocytic replication phase of the parasite, they develop from ring stages to trophozoite and schizont stages in which new merozoites are formed. Interestingly, this intense asexual schizogonic division relies on the levels of host nutrient and more particularly, lipids. Indeed, lysophosphatidylcholine (LPC) present in the host serum is the key limiting nutrient allowing parasite division through provision of important lipid precursor for parasite membrane biogenesis (3,4). Reduction of LPC in cultures in vitro induces parasite conversion from highly dividing asexual merozoites to nondividing sexual gametocytes. During a new blood feed, mosquitos can then ingest stage V gametocytes.

The intraerythrocytic cycle demands the parasite to be able to extend and modify the plasma membrane (PM) and the parasitophorous vacuole membrane (PVM) and to replicate its organelles like the mitochondria and apicoplast, which are both sites of lipid synthesis for the formation of daughter cells. This is a phase of intense membrane biogenesis and current evidence shows that large amounts (5-fold more) of lipids are needed for the survival and replication of the parasite during this asexual cycle (5)(6)(7)(8).

The building blocks of all lipids, the fatty acids (FAs), are predominantly scavenged from the host but can also be fully made de novo via an active prokaryotic type II FA synthase, called FASII, that is present in the relict nonphotosynthetic plastid of the parasite (i.e. the apicoplast). The FASII pathway provides specific FA and is particularly essential during intense schizogonic division of the parasite occurring during the liver-stage and for sporozoite development during mosquito stages (9)(10)(11). However, the activity of the apicoplast FASII is also directly influenced by the level of host nutrient during asexual blood stages, as it can be upregulated and become essential if host nutrient and scavenging are scarce (12,13). Membrane biogenesis is therefore dependent on a combination of host lipid scavenging and de novo parasite lipid synthesis relying on the acquisition of FA building blocks from FASII de novo synthesis and host scavenging (10,11,14,15). Phospholipid (PL) synthesis through assembly of these FA, and trafficking are needed for the generation of membrane compartments for newly formed parasites.

The bulk of PLs, their precursors like the polar heads choline and ethanolamine as well as FA, especially the sole FA species required from the host, C16:0 and C18:1, need to be scavenged from the host serum (10,11,(16)(17)(18). This supports the latest postulate that the parasite is highly dependent on the nutritional status of the host cell and its environment, which has been extensively studied in Toxoplasma gondii, another apicomplexan parasite (19)(20)(21). Currently, this lipid flux in Plasmodium is poorly understood and requires further investigation.

During the lytic cycle, the parasite must be able to sense nutrient changes and adapt to varying metabolic states whereby it can manage a fine balance between lipid storage (neutral lipids) and lyso/PL synthesis required for parasite proliferation or sexual commitment to gametocyte stages facilitating parasite transmission to the mosquito vector (22). Not only is lipid acquisition required, but to maintain lipid homeostasis, PL and other lipid groups must be degraded to generate FA and polar heads, a reaction that is usually catalyzed by phospholipase enzymes (17).

Phospholipases are enzymes which hydrolyze specific ester bonds in PLs and are classified into four groups, A, B, C, and D, based on their hydrolytic activity (17). Over 20 putative phospholipase enzymes are present in P. falciparum and only two have been shown to play a key role in intracellular parasite development, namely phosphoinositide specific-phospholipase C and PfPNPLA2 (23). The latter enzyme belongs to the group of patatin-like phospholipase proteins that are poorly understood thus far. Their putative roles suggested by bioinformatic analyses and databases suggest pivotal and putatively redundant functions in lipid homeostasis at all stages of the development of the malaria parasite (17,24,25). One key question is to determine the function, substrate, and products for each of these phospholipases to unravel the complex lipid metabolism by which the parasite depends for its survival. Here we investigated the phospholipase PfPNPLA2 and compared its expression with two other PfPNPLA proteins, PfPNPLA3 and PfPNPLA4. We reveal their non-overlapping localizations and further elucidate the role of PfPNPLA2 at the parasite mitochondria using lipidomics approaches.

We further demonstrate that PfPNPLA2 has a critical role during low host nutrient content, when the protein becomes essential for asexual blood stage survival, especially at the schizont-to-ring transition. In addition, we show that PfPNPLA2 degrades phosphatidylglycerol (PG) to allow the synthesis of lysobisphosphatidic acid (LBPA). This likely allows the generation of FA for the boost of synthesis for membrane PLs, potentially facilitating parasite division and progeny formation. Our work further emphasizes the importance of mobilization and channeling of lipid resources, here PG degradation/recycling, for asexual blood stage parasite development within the human host RBC.

MATERIALS AND METHODS

Primary antibodies

In this study, the following primary antibodies and antisera were used: Rabbit anti-HA antibody (Sigma-Aldrich); rat anti-HA antibody (Roche); mouse anti-GFP antibody (Sigma-Aldrich); rabbit polyclonal antisera against Pfs230 (Biogenes), Pfs25 (ATCC), Pfs16, AMA1 (26); mouse polyclonal antisera against falcilysin (27), Pf39 (28) and actin I (29). The generation of polyclonal mouse antisera against PfPNPLA2, 3 and 4 is described below (see Sup. Fig. 2C).

Generation of mouse antisera

Recombinant proteins of PfPNPLA2 (spanning aa 571-998), PfPNPLA3

(spanning aa 589-763) and PfPNLPA4 (spanning aa 2-187) were expressed for the generation of antisera. PfPNPLA2 was expressed with an N-terminal maltose-binding protein (MBP)-tag using the expression vector pIH902. Cloning was mediated by the addition of the restriction sites EcoRI and PstI (for primer sequences see Table 1). PfPNPLA3 was expressed with an C-terminal His-tag using pET28c (+) and cloning was mediated by the addition of NcoI/XhoI restriction sites. PfPNPLA4 was expressed as a fusion protein with an Nterminal MBP-tag using pMalc5x. Cloning was mediated by XmnI/PstI restriction sites. Recombinant fusion proteins were expressed using E. coli BL21 (DE3) RIL (Stratagene) and purified via affinity chromatography using amylose resin (New England Biolabs) for the MBP-tagged proteins and cOmplete™ His-tag purification resin (Sigma-Aldrich) for the His-tagged proteins according to the manufacturers' protocols. After PBS buffer exchange via filter centrifugation using Amicon Ultra 15 (Sigma-Aldrich), protein concentrations were determined by Bradford assay. 100 µg recombinant proteins emulsified in Freund's incomplete adjuvant (Sigma-Aldrich) were injected into 6 weeks-old NMRI mice (Charles River Laboratories) via subcutaneous injection. After 4 weeks, a boost with 50 µg recombinant proteins was injected. Ten days after the boost, mice were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture (Sigma-Aldrich)

according to the manufacturer's protocol and immune sera were collected via heart puncture. Sera were pooled from three mice immunized with the same antigen. Experiments for the generation of antisera in mice were approved by the animal welfare committees of the District Council of Cologne, Germany (ref.

no. 84-02.05.30.12.097 TVA).

Generation of the PfPNPLA2and PfPNPLA4-KD parasite lines

PfPNPLA2-and PfPNPLA4-KD parasite lines were generated via single crossover homologous recombination using the pARL-HA-glmS vector (30). A 1015bp gene fragment homologous to the 3' end of the gene coding for PfPNPLA2 and a 840-bp gene fragment of the gene coding for PfPNPLA4 (excluding the stop codons) were amplified using corresponding primers (for primer sequences see Table 1). Ligation of the inserts with the vector backbone was mediated by NotI/AvrII restriction sites. A synchronized wildtype (WT) NF54 culture containing 5 % ring stages was loaded with 100 µg vector in transfection buffer via electroporation as described. Parasites carrying the vector were selected by the addition of WR99210 (Jacobus Pharmaceuticals) in a final concentration of 4 nM and successful integration of the vector was confirmed by diagnostic PCR using (1) 5'-integration primer, (2) 3'-integration primer, (

pARL-HA-glmS forward primer and ( 4) pARL-HA-glmS reverse primer (for primer location see Sup. Fig. 2a; for primer sequences see Table 1).

Diagnostic RT-PCR

Total RNA was isolated from synchronous ring, trophozoite and schizont cultures, as well as from Percoll-gradient enriched immature, mature and activated gametocytes as described before (30). RNA preparation and subsequent cDNA synthesis were performed as described (30). Transcripts for PfPNPLA2 (250 bp), PfPNPLA3 (250 bp) and PfPNPLA4 (250 bp) were amplified in 25 cycles using the corresponding primers (for primer sequences see Table 1). To confirm purity of the asexual blood stage and gametocyte samples, transcript amplifications of ama1 (407 bp) and of ccp2 (286 bp) were performed using the corresponding primers. Amplification of the housekeeping gene aldolase (378 bp) was used as loading control.

Indirect immunofluorescence assay and live cell imaging

Synchronized asexual cultures of a wildtype (3D7) strain and the PNPLA2-iKD line were maintained as previously described (31,32). Parasite cultures (200 µl of compact infected RBS) were collected by centrifugation (5 min at 1 500 rpm at room temperature). Parasites were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde for 30 min at room temperature. Cells were centrifuged at 3000 rpm for 2 mins and the supernatant was discarded. The sample was then washed twice with PBS and the supernatant removed. The pellet was resuspended gently in 1 ml of 1% FBS (fetal bovine serum) in PBS and spun again as previously. Cells were permeabilized using 0.1% Triton X-100 in PBS for 10 mins. Cells were pelleted and the sample was washed twice with PBS. The pellet was saturated with 3% FBS in PBS and incubated at room temperature for 1 h. Thereafter the primary antibodies of HA-tag or anti-PNPLA2/PNPLA3/PNPLA4 were added at dilutions of 1:500 or 1:50, respectively. The antibodies were then washed away twice with PBS. The secondary antibodies (AlexaFluor488/546 anti-rat/mouse) were used at 1:1000 dilutions in PBS and incubated for 1 h. Hoechst stain was used at 1:25000 dilution for 20 mins and finally samples were washed with PBS thrice. Pellets were resuspended in 150 µl of PBS and then gently mixed with the same amount of Fluorogel. 15 µl of each sample was pipetted on a microscope slide and overlayed with a square coverslip. Samples were allowed to solidify in the dark at 37°C for 1 h. Specimens were examined with an Axio Imager; ZEISS, ×100 magnification. Images were treated using ImageJ software. Indirect immunofluorescence images were obtained for gametocytes as previously described (30).

For IFAs using anti-LBPA antibodies (Echelon Biosciences), a dilution of 1:500 was used, and parasites were incubated for at least 1 hour. To determine the corrected fluorescence intensity experiments were performed in triplicate and Image J was used to identify the cell area, mean fluorescence intensity, minimum/maximum fluorescence intensity and integrated fluorescence intensity (33). The same data was then determined concerning an area outside the cell (used as the background). The corrected total cell fluorescence (CTCF)

was calculated with the following equation: CTCF = Integrated Density -(Area of selected cell X Mean fluorescence of background readings)

Western blotting

Western blotting was performed as previously described (30). Briefly, total sorbitol synchronized schizont stage parasite samples underwent saponin lysis to remove red blood cell membrane (34). The sample was then resuspended in a lysis buffer containing an anti-protease cocktail (0.5% Triton X-100, 4%

w/v SDS, 0.5× PBS). SDS-PAGE buffer was added, and samples were denatured at 95°C for 10 mins. Equal sample volumes were then separated by SDS-PAGE and proteins were transferred onto a Hybond ECL nitrocellulose membrane (Amersham Biosciences) according to the manufacturer's protocol.

The blot was blocked using Tris-buffered saline containing 5% w/v skim milk, pH 7.5, followed by overnight incubation at 4°C with polyclonal mouse immune sera specific for Pf39 (dilution 1:5,000) or using polyclonal rabbit immune sera specific to anti-HA (1:1,000). Subsequent washing was followed by incubation with alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich) for 1 hr at RT to reveal the blot. Blots were scanned and processed using the Adobe Photoshop CS software. Band intensities were measured using the ImageJ programme version 1.51f.

Asexual blood stage replication assay

To analyze asexual blood stage replication and length of the intraerythrocytic replication cycle in the PNPLA2-KD parasite line, experiments were set-up as follows: Expression of PNPLA2 was knocked-down in transgenic PNPLA2-KD parasite line by treatment of the culture with GlcN (D-(+)-glucosamine hydrochloride; Sigma Aldrich) at a final concentration of 2.5 mM. GlcN treatment started one cycle before the experimental set-up. The GlcN-treated culture was compared to the untreated culture cultivated in normal cell culture medium. This assay was also done in minimal media only including RPMI, glucose, hypoxanthine (with 1M NaOH), FA-free bovine serum albumin (60 µM) and FFA C16:0 (30 µM) and C18:1 (30 µM) . To exclude an unspecific effect of GlcN, the parental WT line was used as control. For each assay, three experiments were performed, each in triplicate.

Gametocyte development assay

To induce knockdown of PfPNPLA2, a synchronized parasite culture of the PfPNPLA2-KD line was treated with 2.5 mM GlcN for 72 h. Gametocytogenesis was subsequently induced with lysed blood cells while the GlcN treatment was continued. The gametocytemia was evaluated by Giemsa smears over a period of six days. Untreated PfPNPLA2-KD cultures were used as controls.

Lipidomics analysis

Lipidomics analysis was performed on 3-4 independent cell harvests of the PNPLA2-iKD line. Highly synchronous parasite cultures grown for at least 48 hours with or without glucosamine (ring/trophozoite or schizont stage) were harvested by transferring the cultures to pre-warmed 50-ml tubes and the total amount of parasites was determined by Giemsa staining and via counting on a hemocytometer. The cultures were metabolically quenched via rapid cooling to 0°C by suspending the tube over a dry ice/100% ethanol slurry mix, while continually stirring the solution. Following quenching, parasites were constantly kept at 4°C and liberated from the enveloping RBCs by mild saponin treatment with 0.15% w/v saponin/0.1% w/v BSA PBS for 5 min. After three washing steps with 1 ml ice-cold PBS followed by centrifugation each time, the parasite pellet was subjected to lipid extraction using chloroform and methanol (28) in the presence of butylhydoxytluene, PC (C21:0/C21:0), C13:0 or C15:0 FA standards (Avanti). Lipid extraction, separation and analyses were all performed as previously described in Flammersfeld et al., 2020 (30). FA methyl esters were detected by GC-MS (Agilent 5977A-7890B) according to the method described in Ramakrishnan et al. ( 2012) (6) and quantified by Mass

Hunter software (Agilent). Each FA was quantified according to a calibration curve generated with authentic fatty methyl ester standards.

Statistical analysis

Data are expressed as mean ± SD. Statistical differences were determined using unpaired two-tailed Student's t test, as indicated. Values of p < 0.05 were considered statistically significant. Significances were calculated using GraphPad Prism 5 and are represented in the figures as follows: ns, not significant p > 0.05; *p < 0.05; **p < 0.01.

RESULTS

PfPNPLA2 is a non-redundant parasite phospholipase that localizes to both apicoplast and mitochondrion and is important for asexual parasite development under nutrient poor environment Gene PF3D7_1358000 encodes a putative patatin-like phospholipase ( 17), termed PfPNPLA2. The protein is predicted to be a phospholipase A2 (PLA2)

with a patatin-like phospholipase domain like that of PfPNPLA1 (30) and an estimated molecular weight of 238.2 kDa (Fig. 1a). PfPNPLA2 comprises a transmembrane domain at the N-terminal region of the protein. Further, the characteristic GXSXG motif is present in this protein between 1166 aa-1171 aa, as well as a conserved signature catalytic aspartate found in most eukaryotic PLA2, which is found at residue 1201 (Fig. 1A) (35). As previously shown, all plasmodial patatin-like phospholipase homologs share the abovementioned motif and catalytic aspartate, supporting the classification of PNPLA2 (17,30). In addition to PfPNPLA1 and PfPNPLA2, two more members of the PfPNPLA family were previously identified, i.e. PfPNPLA3 (151 kDa, encoded by gene PF3D7_0924000) and PfPNPLA4 (283 kDa, encoded by gene PF3D7_0218600). PfPNPLA3 comprises two N-terminal transmembrane domains in addition to the central patatin-like phospholipase domain. (Sup. Fig. 1A).

Diagnostic reverse transcriptase (RT)-PCR was performed to investigate expression of pnpla2, pnpla3 and Pfpna4 in the P. falciparum blood stages and the synthesis of transcripts in the ring and trophozoite stages and schizonts were demonstrated for all three genes.. Similar transcript levels were detected in immature (stage II-IV) and mature (stage V) gametocytes as well as in were generated against recombinant PfPNPLA2, PfPNPLA3 and PfPNPLA4 peptides to be used for protein expression analysis. Immunofluorescence assays (IFA) revealed that PfPNPLA2 is expressed throughout all asexual and sexual blood stages (Fig. 1C). PNPLA2 is predominantly expressed in the schizont stages and localizes to vesicular structures of individual merozoites formed within the parasite vacuole. PfPNPLA2 could further be detected at distinct loci within the cytoplasm of gametocytes (Fig. 1C). Closer colocalization studies revealed that PfPNPLA2 does not colocalize with PfPNPLA3 or PfPNPLA4 (Fig. 1D-E). We performed further co-localization IFA, using an apicoplast-tagged parasite line, the Plasmodium falciparum apicoplast triose phosphate transporter TPT-HA line (12,38), with antibodies against native PfPNPLA2. This revealed that PfPNPLA2 colocalizes with the apicoplast marker, especially during ring and trophozoite stage (Fig. 1F). However, IFA using Mitotracker™ also revealed that, in some rings to schizont-stage parasites, this protein largely localizes to the mitochondria. This supports previous data showing mitochondrial association (23). Our data points at a dual localization and suggests a potential dual/complementary function around/within the apicoplast and the mitochondrion (Fig. 1G).

To determine the importance of PfPNPLA2, we performed growth assays upon presence or absence (+GlcN) of the protein. The reduction of PfPNPLA2 in the mutant line by treatment with 2.5 mM GlcN revealed no significant effect on intraerythrocytic replication during a period of 72 h in regular in vitro culture conditions, when compared to untreated PfPNPLA2-HA-KD parasites and GlcN-treated or untreated WT NF54 parasites (Fig. 2A). In addition, quantification of ring, trophozoite and schizont stages at different time points did not reveal a major effect of GlcN-treatment on parasite stage development of PfPNPLA2-HA-KD and WT NF54 parasites (Sup. Fig. 3), suggesting that the reduction of PfPNPLA2 does not delay the blood stage cycle. (Fig 2A).

According to likely predicted essentiality scores for the protein in a piggyBac study of the P. falciparum genome, these data were surprising (39). It could be that the incomplete reduction of protein expression was sufficient to allow for parasite survival.

We previously showed that the host nutritional and lipid content can influence the essentiality of a single protein, such as one protein can be non-essential in normal/rich lipid conditions, while becoming essential under scarce nutritional conditions (13). The mild phenotype due to incomplete protein expression reduction prompted us to set up an experiment to analyze this. Parasites were cultivated in minimal media (11) containing the minimal FA requirement for in vitro culture, i.e. C16:0 and C18:1 (in combination to FA-free BSA).

Interestingly, in these lipid-limiting conditions, the PfPNPLA2-HA-KD line displayed a significant growth delay after 96 hours of intracellular development.

This led to a decrease in ring stage parasites (Fig. 2B). In sexual stage parasites, the depletion of this protein did not affect the gametocyte population (Fig. 2C). These data may suggest that PNPLA2 is more important for asexual blood stage development than for gametocytogenesis, and the essentiality of this protein cannot be ruled out, as we were only able to achieve 50% protein reduction. More importantly, this protein seems to be important under nutrient poor conditions and may strongly be dependent on the extracellular environment of the host.

This also correlates to our findings that growth defects following PfPNPLA2 knockdown occur under low lipid content only containing C16:0 and C18:1.

However, no changes could be detected in the FA profiles of TAG or total PL levels (Sup. Fig. 4A-B) under PfPNPLA2 deficiency.

Knockdown of PfPNPLA2 in trophozoite stage parasites exhibited limited changes in the overall FA profile of the total lipid content of the parasite (Fig. 3E), namely a significant increase in C18:0 and a significant decrease in C18:1.

Further analyses of parasite lipid classes showed no changes in total neutral lipid content (either FFA, DAG or TAG) and PL content (Fig. 3F). This lack of phenotype might be directly linked to a lower activity of the enzyme during trophozoite stages as its RNA expression is the lowest during trophozoite stages (25).

The most severe effects were observed during schizont stages when the protein is most expressed. First, in the total lipid FA profile, the loss of PNPLA2 in schizont stage resulted in a significant decrease of the relative abundance of palmitic acid (C16:0) (Fig. 3G). Similarly, as in ring stages, knockdown of PfPNPLA2 in schizonts resulted in a significant increase of the storage lipid, TAG. However, unlike in ring stages, the levels of both FFA and DAG were significantly reduced in schizonts (Fig. 3H), but there were no changes in their FA species profile (Sup. Fig. 4C-D). The reduction of PfPNPLA2 levels did not lead to any changes in the relative amount of total PL levels (Fig. 3H). Analysis of the FA content of TAG accumulating in schizont stage in the absence of PfPNPLA2 revealed a significant increase of TAG molecular species containing C16:0 and C18:1 (Fig. 3I), as previously observed in rings, suggesting increased host scavenging. It should also be noted that there were no changes in the total lipid abundance at any stage, despite seeing clear changes in individual lipid groups (Sup. Fig. 4E).

These data collectively suggest that PNPLA2 is most important for FFA generation/acquisition as well as the synthesis of neutral lipids likely for storage and timely mobilization during the transition from the schizont to the ring stage parasites.

PfPNPLA2-deficiency results in an overall increase of PG and decrease of

LBPA

To further investigate the putative phospholipase activity of PNPLA2, notably determine its potential lipid substrates and products, we sought to analyze each PL class and their molecular content in schizonts by 1D or 2D HPTLC coupled to GCMS lipidomic analyses.

The knockdown of PfPNLA2 significantly impacted parasite PL homeostasis (Fig. 4A). There were slight, yet significant reductions in the content of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) that seemed more marginal. PLA2 usually catalyze the hydrolysis of one of the FA moieties from PL, hence generating lysoPL and FA. The levels of major lysolipids, lysophophatidylcholine (LPC), and lysophosphatidic acid (LPA) were not significantly impacted in the absence of PfPNPLA2, suggesting they might not be the direct products of the enzyme.

The most striking change, however, was a significant increase of phosphatidylglycerol (PG). PG is usually a low represented class of membrane PL that is also the substrate for the essential de novo synthesis of the mitochondrial-signature lipid class, cardiolipin. However, no change was observed in the levels of cardiolipin (CL) in the absence of PfPNPLA2, again suggesting CL not being the product of the phospholipase. Interestingly, PG can also be the substrate of PLA2s to generate lysophosphoglycerol (LPG), or LBPA/bismonoacylglycerolphosphate (LBPA/BMP), both of which are important lipid precursors/intermediates also present in apicomplexa in low amounts (13,(40)(41)(42). LBPA/BMP can also be made from cardiolipin but the usual substrate in mammalian cells is usually PG (40). We thus sought to quantify the levels of LPG and LBPA and determine their potential change in content upon the disruption of PfPNPLA2 (Fig. 4B). Our approach thus reveals that upon the loss of the enzyme, there was a significant accumulation of PG together with a very significant decrease of LBPA, whilst LPG levels were not significantly impacted (though slightly reduced). This significant decrease in LBPA was further confirmed by immunofluorescence (using an anti-LBPA antibody), which showed a significant decrease in fluorescence within the parasite when the protein expression was inhibited by GlcN treatment (Fig. 4C-D). Taken together, the data strongly suggest that PfPNPLA2 is responsible for the degradation of PG to form LBPA, which further induces PL homeostasis disruption (see hypothesis model in Fig. 5).

DISCUSSION

Phospholipases contribute to the virulence of many pathogens, and more specifically to Plasmodium parasites (30). This family of proteins is pivotal for PL recycling, which direct a diversity of physiological roles via the regulation of lipid homeostasis for membrane synthesis but also through the control of key signaling pathways that govern host egress, secretion and developmental switching (3,17,30,(43)(44)(45). Knowing the importance of lipids, membrane biogenesis and homeostasis for the development and survival of P. falciparum, the enzymes manipulating lipids must play central essential parts.

Phospholipases are such enzymes as they can hydrolyze membrane PL, either existing from the host or not, to enable the release of FA and LPL that can be used for parasite membrane homeostasis. Interestingly, phospholipases rare metabolic genes located in the sub-telomeric region of P. falciparum genome, potentially favoring gene duplication as for var and rifin surface antigens, reflecting their putative importance for the parasite (46). Their unusually high numbers (at least 22 putative phospholipase genes) compared to typical eukaryotes also reflect their importance for P. falciparum (17,45). Their exact roles for the parasite remain poorly understood but recent studies have started revealing differential localization and non-redundant and major functions such as hemozoin formation, parasite division or gametocyte formation (30,44,47,48). The few that have been investigated also show potential as drug targets (49,50). To date however, the exact functions and mechanisms of most phospholipases remain to be unraveled.

As with PC and LPC, PL and lysoPL balances in the parasite can govern their cell development and are at the mercy of phospholipase (3,17). This study focused on a putative phospholipase enzyme containing a patatin-like phospholipase domain (Fig. 1A). We generated an inducible knock-down line for PfPNPLA2 (PF3D7_1358000) using the glmS-ribozyme system (51), but were unable to achieve complete protein degradation. This could suggest why we did not see a decrease in parasite viability as previously described in [START_REF] Burda | Global analysis of putative phospholipases in the malaria parasite Plasmodium 1 falciparum reveals critical factors for parasite proliferation 2 3[END_REF]. We revealed that PfPNPLA2 also seems to be important for parasite replication in asexual parasites under nutrient stress, specifically when exogenous lipids are limited (Fig. 2A). This seems to be in accordance to the essentiality data from a genome-wide saturation mutagenesis screen in P.

falciparum (52).

PNPLA1 (PF3D7_0209100), has been previously identified as critical for gametocyte induction (30) and the process of gametocytogenesis itself (48,49). PfPNPLA2-HA-tagged parasites in this study have been able to prove dual/proximal localization of this protein to both the mitochondria and the apicoplast of the parasite. This is supported by the predicted mitochondrial targeting sequence found in N-terminal end of the protein (48). These data correlate to the decreased transcription of this protein in gametocytes and could suggest this protein is not critical during sexual blood stages (25).

Localization of the enzyme to the apicoplast might be explained by the close vicinity and the shared metabolic pathways between the this organelle and the mitochondrion (53,54). Currently are there is no known shared lipid metabolism pathways between the two organelles, even if this could be occurring, notably for potential lipid exchange between them. The T. gondii homologue of PfPNPLA2 (TgPL2) does solely localize to the apicoplast and is involved in apicoplast biogenesis and parasite lipid homeostasis, more precisely by affecting levels of PC and LPC (55). The proximity to the mitochondria suggests that PfPNPLA2 could be involved in mitochondrial membrane biogenesis through the generation of FA and lysoPL precursors. We hypothesized that this could be linked to cardiolipin synthesis as it only occurs at the mitochondrial level [START_REF] Aho | REACTIVITY OF VARIOUS ANTICARDIOLIPIN ANTIBODIES WITH INTACT MITOCHONDRIA[END_REF]. The full cardiolipin synthetic pathway is yet to be characterized in P.

falciparum, but in other eukaryotes, it requires PG as a substrate [START_REF] Gallala | Biological function of the cellular lipid bmp-bmp as a key activator for cholesterol sorting and membrane digestion[END_REF].

Lipidomics revealed that PG was the most strikingly affected PL class in the absence of PfPNPLA2, yet without significantly affecting cardiolipin levels. The disruption of the protein resulted in the accumulation of PG instead, unlike in the knockout of the T. gondii homologue of PfPNPLA2, which displayed increased levels of PC (55). However, PG, and or LBPA can be degraded Another important point is that the most significant growth phenotype occurred in low host lipid environment. We and others showed that host nutrient levels directly influence parasite metabolic activity, especially lipid metabolism, which can be rewired/adapted by the parasite upon host environment (3,19,24,[START_REF] Mancio-Silva | Nutrient sensing modulates malaria parasite virulence[END_REF][START_REF] Walsh | Toxoplasma metabolic flexibility in different growth conditions[END_REF].

Indeed, de novo lipid synthesis and host scavenging activities can be used alternatively to compensate one another (13,19). Host lipid scavenging constitutes the major source of parasite lipids and can be upregulated if de novo synthesis is insufficient. Scavenged lipids, specifically their FA moieties, are usually channeled towards lipid storages, usually lipid droplets, in the form of TAG, which can be timely mobilized for membrane biogenesis during parasite division (19,50). Here, we see that disrupting PfPNPLA2 affects parasite growth only under low host lipid environment, indicating that the enzyme likely plays a role to boost lipid synthesis when scavenging is scarce (Fig. 5). This is further correlated by the increase in C16:0 and C18:1 that are accumulating in TAG during schizont, and ring stages in the absence of PfPNPLA2 in high host lipid content. This suggests that the parasite increases scavenging and lipid storage to compensate for the loss of PfPNPLA2, as previously observed for the loss of another P. falciparum lysophospholipase, PfLPL3 (50). This is further correlated by the additional accumulation of all neutral lipids, TAG, DAG and FFA during ring stages, reflecting the accumulation of scavenged material normally channeled through the storage lipid synthetic pathway, as previously observed (19,50).

One possible hypothesis (Fig. 5) for the reduction of parasite population in the absence of PfPNPLA2 under low host lipid content is that, similarly as in the PfLPL3 mutant, the parasite does not have enough supply of FA by the degradation of its lipid substrate, in this case, PG. This, in return, could prevent the parasite lipid synthesis machinery to form PLs for parasite membrane biogenesis during division, hence reducing progenies and overall parasitaemia. This is further supported by the significant decrease of major PL classes PC, PE, PI and PG when PfPNPLA2 is absent (Fig. 5). A potential explanation is that the degradation of PG can provide FA moieties for the synthesis of major lipid classes when host lipid resources are scarce, hence enabling optimal progeny formation even under sub-optimal physiological conditions. phosphatidylinositol). In the absence of PfPNPLA2 PG levels are increased with lowered levels in LBPA and FFA. We hypothesize that the lack of sufficient FFA prompts the parasite to increase host FFA scavenging of C16:0 and C18:1. This does not restabilize FFA to normal and instead TAG formation (LD: lipid droplet) is favored to escape lipotoxic death. The increase in PG has a negative feedback effect and PL synthesis (PC, PI, PE) is inhibited. schizonts (SZ), as well as immature gametocytes (imGC; stages II-IV), mature gametocytes (mGC; stage V) and activated gametocytes (aGC; 15' p.a.). Transcript analysis of ama1 (407 bp) and ccp2 (286 bp) were used to demonstrate purity of the asexual and sexual blood stage samples. Transcript analysis of aldolase (378 bp) was used as loading control, RNA samples lacking reverse transcriptase (-RT) were used to prove the absence of gDNA. For transcript expression of Pfpnpla1, see [START_REF] Flammersfeld | A patatin-like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum[END_REF] integration of the pARL-HA-glmS vector. Diagnostic PCR using gDNA of the PfPNPLA2-HA-KD and the PfPNPLA4-HA-KD lines demonstrates successful 5′ (primers 1 and 4; 1,182 bp for PfPNPLA2-HA-KD; 1,129 pb for the PfPNPLA4-HA-KD) and 3′ (primers 2 und 3; 1,575 fpr PfPNPLA2-HA-KD; 1,166 bp for the PfPNPLA4-HA-KD) integration. As a control, amplification of WT NF54 gDNA was aimed (primers 1 and 2; 1,501 bp for the PfPNPLA2-HA-KD; 1,205 bp for the PfPNPLA4-HA-KD).

TABLES

FIGURES AND FIGURE LEGENDS

Episomal DNA was further detected (primers 3 and4; • In this study we also show that the deletion of this protein and a paralogue protein, TgDGAT1, leads to an increase in parasite development suggesting that these proteins play a collective role in controlling parasite progression

• The loss of these proteins both result in an accumulation of free FAs that cannot be 'detoxified' by incorporating them into LDs, leading to parasite death.

• The essentiality of these two proteins was nutrient dependent showing that the parasite can reprogram its metabolic activity and adapt to a changing host environment In summary, we identified that the first esterification step of the ER glycerolipid pathway forming LPA is crucial for parasite propagation. We also show that LD biogenesis/metabolism is crucial under low nutrients as these storage moieties serves as indispensable deposits of fuel for parasites. Finally, the nutrientdependency of these proteins provides insight into the parasite's nutrient flexibility, in that they can adapt to different environmental conditions rendering proteins that were once non-essential, essential, or vice-versa.

Résumé du manuscript:

Ce manuscrit décrit mon dernier projet de thèse, qui vise à caractériser le rôle d'acyltransférases putative, la glycérol-3-phosphate acyltransférase (TgGPAT) et la diacylglycérol acyltransférase 2 (TgDGAT2). Ce projet renforce la thématique de flexibilité métabolique retrouvée chez Toxoplasma gondii abordé au cours de ma thèse. Nous avons développé des lignées parasitaires permettant l'expression inductible des protéines mentionnées pour étudier leur activité dans des conditions nutritives variées.

Les résultats principaux sont les suivants :

• TgGPAT est la protéine eucaryote homologue à la protéine TgATS1 retrouvé dans l'apicoplaste.

• TgDGAT2 n'est pas caractérisée mais diffère de TgDGAT1

• Ces deux protéines sont associées au réseau endomembranaire du parasite

• TgGPAT est essentielle au développement du parasite et permet la formation d'acide lysophosphatidique qui joue un rôle clé dans la biogenèse des membranes et des lipides neutres.

• TgGPAT est essentielle en conditions de stress nutritive et en situation d'excès nutritif, à l'inverses des autres acyltransférases comme TgAGPAT or TgATS2

(qui sont responsables de la seconde étape d'estérification des acides gras au squelette de glycérol-3-phoshate pour former des acides phosphatidiques).

• Ces observations suggèrent que le rôle catalytique des GPATs (TgGPAT and TgATS1) gouverne une réaction indispensable à la survie du parasite.

• TgDGAT2 en revanche semble seulement importante en conditions de stress nutritif est permet la formation de gouttelette lipidiques.

• Dans cette étude, nous montrons également que la délétion de cette protéine et de son paralogue TgDGAT1 favorise le développement du parasite, suggérant un rôle conjoint dans le contrôle de la croissance parasitaire.

• La délétion de chacune de ces protéines mène à l'accumulation d'acides gras libres qui ne peuvent pas être incorporés dans les gouttelettes lipidiques et deviennent toxiques pour le parasite. acyltransferase (TgGPAT) and diacylglycerol acyltransferase 2 (TgDGAT2) that localize to the endomembrane system and cytosol of the parasite. We emphasize the nutrient role of these proteins as in different lipid environments the parasite can render a protein that was once non-essential, essential, and vice versa. TgGPAT is a critical protein needed for parasite development. Lipidomic analyses showed this protein is responsible for lysophosphatidic acid formation, which is pivotal to membrane biogenesis and neutral lipid synthesis. Lipid droplet (LDs) IFA also indicated that this protein has a role in TAG formation, the main constituent of LDs. TgDGAT2 is a nonessential cytosolic protein that is involved in LD biogenesis as confirmed by IFA.

Lipidomic approaches revealed that its disruption impacted the lipidomic profile of the parasite, specifically DAG levels. This study revealed that these proteins are essential for glycerolipid metabolism and act in a regulatory manner in different nutrient conditions. The loss of these proteins both result in an accumulation of free FAs that cannot be 'detoxified' by incorporating them into LDs, leading to parasite death.

Introduction

Apicomplexa parasites, such as Plasmodium falciparum and Toxoplasma gondii, are unicellular eukaryotes responsible for major human diseases affecting about a third of the global population. Traditionally toxoplasmosis is not life threatening but can be even lethal to those suffering from immunological impairment, cancer, AIDS, and organ transplant recipients (1,2) No vaccine or efficient drug exist to treat this disease.

Current evidence shows that lipid synthesis and trafficking is pivotal for parasite-host interactions and critical for parasite survival, hence representing a pertinent drug target (3,4).

Parasites need a large amount of lipids for the generation of membrane compartments for parasite propagation and pathogenesis within the human host. Fatty acids (FA) are the building blocks of lipids and are acquired by the parasite either through the FASII pathway (de novo) or by directly scavenging them from the host and host environment (5,6). These FAs are then channelled into phospholipid (PL) synthesis for membrane biogenesis or storage as lipid droplets (LD) for timely controlled usage and parasite survival (7,8). Toxoplasma tachyzoite parasites exemplify the need for new membranes to maintain parasite propagation. This is achieved by the continuous FA synthesis and scavenging (8). We recently identified a nexus protein, TgLIPIN, that is responsible for channelling FAs to either membrane biogenesis or lipid storage.

The main metabolites of these pathways lysophosphatidic acid (LPA), phosphatidic and phosphatidylinositols (PIs) (7). LPA/PA can be further catabolized to for storage lipids like diacylglycerol (DAG) and triacylglycerol (TAG). DAG can be directed through the Kennedy or CDP-DAG pathway for PL synthesis, or it can continue through the glycerolipid pathway and be esterified to from TAG, the main constituent of LDs. These pathways are governed by nutrient availability in terms of the physiological environment of the host cell (12)(13)(14)(15). The fate and incorporation of these FAs into PLs or LDs are reliant on the activity of specific acyltransferase (ATs). There are many different ATs identified and studied (16)(17)(18)(19)(20)(21)(22)(23)(24)(25), the ATs responsible in determining the fate of fatty acids to PLs or LDs upon the response to the nutrient availability are yet to be determined.

One of the first acyltransferase involved in glycerolipid precursor synthesis is named glycerol-3-phosphate acyltransferase (GPAT (16). It facilitates the rate limiting reaction involving the conversion of glycerol-3-phosphate (G3P) to LPA by esterifying a fatty acyl-CoA (FA) to the sn-1 position of the G3P backbone. Then to add another fatty acid to the LPA, there is another AT, Acylglycerol-3-phosphate acyltransferase (AGPAT). This enzyme is responsible for adding the second activated acyl-CoA to LPA on the sn-2 position of the G3P backbone, forming PA. In eukaryotic organisms, many isoforms of GPAT/AGPATs exist with different cellular localization and function.

GPAT1/2 in humans are mitochondrial and seem to modulate fatty acid oxidation (16).

GPAT3/4 in most models are found near the ER (16). GPAT3 in humans has a controversial identity as it seems to function as both a GPAT and AGPAT (26).

Thereafter another group of ATs exist, which transfer an activated FA to sn-3 position of G3P. Since sn-3 position is usually allocated to the head group of phospholipids, these ATs are specific to DAG to generate TAG, i.e. diacylglycerol acyltransferase (DGAT). Both TAG and DAG can be then used as energy storage and/or of FA to fuel the phospholipid synthesis machinery (27). DAG to be acylated can be de novo synthesized from PA by the removal of its phosphate group by Phosphatidic acid phosphatase or LIPIN (7).

In most eukaryotic models, at least two DGATs coexist and contribute to synthesis and metabolism of TAG (18,28). DGAT1, which is part of the membrane-bound Oacyltransferases or MBOAT super family) is typically more involved in the recycling of hydrolysed TAGs by re-esterifying FAs (29). This enzyme may also be important for preventing lipotoxicity and protecting the cell from highly levels of FAs, having a preference to exogenous FAs. In contrast to DGAT1s, the model of DGAT2 suggest this protein is instead more active when FA levels are low. This enzyme is frequently associated to endogenous/de novo FA synthesis and esterification (17). In terms of sequence motifs, DGAT1s are more conserved than DGAT2s(30).

In T. gondii parasites several ATs have been identified and characterised. Previously, we identified an apicoplast-derived GPAT (TgATS1) and TgAGPAT (TgATS2) as well as an ER-localised TgAGPAT (24,25). TgATS1 is an essential protein and is lethal when knocked out, unlike TgATS2 (31). Both TgATS1 and TgATS2 are responsible for the bulk lipid synthesis using FAs from the FASII for rapid membrane biogenesis supporting parasite survival (31,32). These enzymes are also identified and partially characterised in Plasmodium spp. (32,33). In P. yoelli ATS1 has shown to be essential for liver stage parasites (34). A GPAT in P. falciparum has also been identified but not characterized lipidomically (35). In T. gondii a DGAT, TgDGAT1, was found not to be essential in this parasite. Its disruption exhibited a decrease in LDs and an accumulation of palmitic acid (C16:0). TgDGAT1 may additionally have a role in the ancient eukaryotic type of diacylglycerol esterification in Apicomplexa parasites (36).

The roles the above-mentioned ATs in the transfer of host-scavenged FAs is not known. Moreover, we do not understand how Apicomplexan parasites can orchestrate continuous bulk PL synthesis and lipid storage under constant FA scavenging which is dependent on the nutrient environment.

To investigate how the parasite uses or stores FA, through the synthesis of LPA/PA and TAG, respectively, we then searched for putative GPATs and DGATs. In T. gondii, there are still uncharacterised ATs such as GPAT (TGGT1_256980, phenotype score = -2.83, (37,38), and DGAT2 (TGGT1_275600, phenotype score = 0.35, (37,38). It is possible that these acyltransferases maybe involved in the transfer of FAs in the response to the nutrient condition. Here, we identified TgGPAT and TgDGAT2 to study their role in the synthesis of TAG under different lipid environments. IFA analyses show these proteins either in proximity with the endomembrane system or cytosol of the parasite. Viability surveillance showed that the TgGPAT is critical for parasite survival, whereas TgDGAT2 seems to be less crucial but still affects the lipid metabolism according to our lipidomic analyses. Our metabolic labelling and massspectrometry-based experiments revealed these proteins play important roles in PL and TAG synthesis that are highly governed by the host nutrient environment.

Material and Methods

Sequence analyses of TgGPAT and TgDGAT2

Sequence alignments were generated using ClustalW using predicted protein sequences of GPAT3 and DGAT2 subfamily proteins from different species namely:

Homo sapiens GPAT3 (Q53EU6), Saccharomyces cerevisiae GPT2 (P36148), (NCLIV_029090) and Hammondia hammondi DGAT2 (HHA_275600). Secondary structure and putative residues are indicated on ToxoDB (37). Putative transmembrane domains were predicted using Deep TMHMM (39). Consensus sequences were found using work by Cao et al., (2011) and Yamashita et al., (2014) (26,30).

Saccharomyces

3D-structure modelling was done by threading the sequence of TgGPAT in the online software Swiss-Model (https://swissmodel.expasy.org/), which lead to the identification of the crystal structure of CmATS1 (40). The sequence surrounding the 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 (1:2000, Life technologies) for 15 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 (Axioimager, Zeiss).

Nile Red staining of lipid droplets

The parasites were allowed to infect and grow in confluent monolayer HFF grown on coverslips, with and without auxin for 24 h. These coverslips were prepared as above and then incubated for 1 h with nile red in 1X PBS before proceeding with DNA staining with Hoechst. For visualizing nile red stained droplets yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) ( 41) was used on the axioimager. Quantification was preformed based on iterative image processing of parasites with and without auxin in differential FBS conditions (0/1/10%) (42).

Western-blot analysis

Parasites were harvested for western blot analysis following complete egress from their host. 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 numbers. Equal amount of protein was separated on a 4-12% gradient SDSpolyacrylamide (Life Technologies) and transferred to Nitrocellulose membrane using the XCellII Blot Module (Invitrogen). Primary antibodies anti-HA (Rat, Roche) and anti-TOM40 (Rabbit) (Van Dooren et al. 2008) were used at a dilution of 1:200 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 characterization:

Plaque assays in Tg mutants

The extracellular parasites were harvested after filtration and counted by haemocytometer. Mutant parasites (~1000) were used to inoculate confluent HFF flask (25 cm 2 ). The mutant parasites were grown for presence or absence of auxin (100 µM) for 7-10 days. After aspiration of the media cells were fixed with 100% ethanol for 5 min and plaque sizes were visualized by crystal violet staining (30-60 min) followed by 3

washes with phosphate-buffered saline. The average plaque areas (particles) were analysed using ImageJ software (n=3 independent experiments).

Replication assay in Tg mutants

In 4-well plates (with coverslips) equal numbers of parasites were allowed to invade confluent HFF cells for 2 h. After invasion, the plates were washed (X3) with ED1

(DMEM, 1% FBS) and parasites were grown for 24 h with or without auxin (100 µM).

These coverslips were then processed for IFA and the number of parasites per vacuole were calculated (n=3 independent experiments).

Lipidomic analysis:

Mutant parasites were grown on confluent monolayer HFF cells in 175 cm 2 flasks until host cells were fully lysed and then reinoculated and grown for a further 24 h with or without auxin (100 µM). Parasites were harvested as intracellular tachyzoites (at least 1 × 10 8 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 4⁰C. The parasite pellet obtained was washed with ice-cold PBS (X3), before transferring the final pellet to a microcentrifuge tube. GC-MS sample preparation and analyses were performed as described in Shunmugam and Dass et al., (2021).

Then total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v) containing PC (C21:0/C21:0 at 10 nmol) and C13:0 or C15:0 (10 nmol) as internal standards for extraction. 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 1 µl butanol/10 7 parasites.

Total lipid analysis:

The extracted total lipid sample was then added with C15:0 or C13:0 (1 nmol), as an internal standard as stated before using TMSH for total fatty acid content. Resulting fatty acid methyl esters (FAME) were analysed by GC-MS as previously described (31,43). 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.

Phospholipid and neutral lipid analysis:

For phospholipid analysis, the extracted total lipid extracted (as above) was separated with 1 nmol LPA (C17:0) or PA (C17:0/C17:0) (Avanti Polar lipids) by two-dimensional silica gel high-performance thin-layer chromatography (HPTLC, Merck). The solvent system used for the 1st and 2nd 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 scraped 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 85 °C for 4-4.5 h. The resulting FAMEs were extracted with hexane and analysed by GC-MS (Agilent).

Lipid flux analyses via stable isotope metabolic labelling combined to lipidomics:

Tracking apicoplast FASII fatty acids:

The mutant parasites were infected to a 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 auxin (100 µM). The parasites were harvested up to 24 h post depletion and lipid were extracted 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 C-labeled fatty acids was obtained by calculating the concentration of all isotopomers of 13 C-labeled FAMEs and finally normalizing to authentic internal standards and parasite number.

Tracking host-derived fatty acids

The HFF cells were grown to confluency in the presence of stable isotope U-13Cglucose at a final concentration of 800 µM added to a glucose-free DMEM (1% FBS).

After harvest and lipid extraction, similar calculations were performed as explained for FASII-derived lipids.

Statistics and reproducibility:

The graphical data for this study was generated using GraphPad Prism software.

Three biological replicates were used per experiment (at least n = 3, unless stated otherwise). The error bars are representative of the standard error of mean (SEM) for each study. Statistical significance was deter-mined for each experiment by unpaired t tests using GraphPad Prism. The range of statistical significance was signified as per the p value indicated in each graph.

Results

Identification of a GPAT3 and a DGAT2 in Toxoplasma parasites

Previously, we identified plant-like acyltransferases, TgATS1 and TgATS2 and an ERlocalised TgAGPAT in the T. gondii genome (31,32). In addition, there are at least 4 other putative acyltransferases encoded in the T. gondii genome (44). To investigate ATs involved in the use or storage of FA, through the synthesis of LPA/PA and TAG, respectively, we searched for putative GPATs and DGATs amongst these putative candidates. Previous studies (31,32,37) and further searches on ToxoDB (37) revealed the presence of an animal-eukaryotic-like GPAT (TGGT1_256980) and a DGAT (TGGT1_275600). Protein sequence analyses by CLUSTAL multiple sequence alignment using the software MUSCLE (version 3.8) ( 45) was performed and uncovered that these proteins fall under the GPAT3 and DGAT2 subfamilies of ATs, respectively (Figure ).

The putative TgGPAT (1002 aa, with 4 transmembrane domains) contained all 4 conserved motifs of this type of protein. The signature catalytic domain for GPATs is found in motif I (HXXXXD) and seems to also be conserved in other apicomplexans.

Phylogenetic analyses of multiple types of GPATs (Fig. 1A) revealed that TgGPAT is like GPAT3-like ATs and clusters with the only two GPATs that exist in yeast. All motifs were predicted (Deep TMHMM) to be on the cytosolic side of the ER membrane like its human homolog HsGPAT3 (16,26,39). As with other sequence studies, motif III and motif II of this enzyme family is the most and least conserved, correspondingly within apicomplexa models (16). Structure modelling of TgGPAT using Cucurbita moschata (squash or pumpkin) ATS1 (CmATS1, ID: Q9FEQ0) structure ( 40) as a threading template suggest that TgGPAT contains the putative G3P binding domain found in motif III (Fig. 1B). Only the region surrounding motif III seemed to align with the crystal structure of CmATS1. No membrane-bound AT crystal structure is available yet to preform structure modelling (46). This can explain the poor alignment to the other 4 motifs when designing this model, since ATS1 is plastid-derived and soluble. From these bioinformatic analysis, TgGPAT is predicted to be a putative AT required for the transfer of FA to form LPA in the parasite ER.

TgDGAT2, as expected showed similarity with DGAT2 type enzymes (30). TgDGAT2 contains all DGAT2 motifs (I-VI) and has conserved amino acid sequence as seen in Cao (30). The most conserved regions include motif I, II and IV. Phylogenetic analyses shows that TgDGAT2, as expected, clustered with other putative DGAT2 proteins in apicomplexa and is closely related to mammal and plants DGAT2 (Sup Figure 1C).

Unlike the other apicomplexa proteins, the orthologue of TgDGAT2, a putative P.

falciparum DGAT, did not align with the consensus sequences of DGAT2 and instead showed similarity to DGAT1 (47). Since no crystal structure has been published a DGAT2 family protein, putative 3D modelling of TgDGAT2 was not preformed (46).

TgGPAT is essential for parasite growth and replication during tachyzoite stages in all nutrient conditions

To determine the importance and function of TgGPAT and TgDGAT2, inducible KD parasite lines of TgGPAT and TgDGAT2 were generated using auxin-inducible-degron system. These lines were generated with a C-terminal HA-tag in the Tir-RH parental line (48). The presence of the HA-tag and its locus were confirmed by PCR (Sup. Fig 2 and3).

We first confirmed the TgGPAT inducible knockdown strain. Western blot analysis showed the TgGPAT-mAID protein migrated at its expected size of 110 kDa (Fig. 1A)

and confirmed the downregulation of TgGPAT-mAID in the presence of 100 µM auxin within 30 min of incubation (Fig. 1A). Immunofluorescence analyses (IFA) revealed that TgGPAT-HA signal localized to the cytosol, and more particularly to the periphery of the nucleus. Characteristic of a GPAT3-type AT, that localizes to the ER, we wanted to confirm if this was the case for TgGPAT. By performing a co-transfection with Der1-GFP, we found that TgGPAT partially localized to the ER and cytosol (Fig. 1B).

To determine the effect of the TgGPAT loss in the parasite's growth, plaque assays was performed. Furthermore, we wanted to determine if the nutrient availability played a role in the proteins function over parasite survival, so we preformed this viability assay at varying FBS (foetal bovine serum) conditions (0-1% FBS). In the normal media condition, 1% FBS, there was already a significant decrease in the growth (Figure C, middle). Plaque assays performed in 0% and 10% FBS medium also demonstrated a significant decrease in parasite growth (Figure C, left and right).

However, as the FBS content increased (0% to 10%) the difference in plaque area between treated and untreated cultures lessened, suggesting that even in the absence of TgGPAT the large availability of lipids from the host's environment allows the parasites to partially compensate. These data suggest that the role of TgGPAT is strongly associated to nutrient availability. The growth defect was also supported by a replication assay which showed a significant increase in small-numbered parasite vacuoles (1-2 parasites par vacuole) and a decrease in larger vacuoles (3-10 parasites par vacuole), which suggested impaired parasite development in the absence of TgGPAT (Figure D).

TgDGAT1 and TgDGAT2 are important to parasite growth under lipid starvation

We then characterized the TgDGAT2-mAID inducible knock down strain. Western blot analysis showed that the TgDGAT2-mAID protein migrated around 60 kDa (Figure 2A), just above its loading control TOM40 (40 kDa). By western blot, we confirmed the complete protein degradation after 24 h under 100 µM auxin treatment. IFA showed that TgDGAT2-mAID fluorescence localizes to the cytosol throughout the parasite cell.

Under auxin treatment, TgDGAT2-mAID signal was not observed throughout the parasite cytosol, as expected, however a remaining fluorescent signal was observed condensed at or in close vicinity to the apicoplast CPN60 signal (Figure B,CPN60).

This could suggest incomplete degradation of the protein, or a background signal seen from florescence in the apicoplast.

To determine the effect the loss of TgDGAT2 protein had on parasite growth, a plaque assay was performed in different nutrient conditions in the same way as for TgGPAT (Fig. 3C). The loss of TgDAGT2 by auxin treatment only led to a significant deficit in parasite progression when lipids/nutrients were retrained, specifically 0% FBS (Figure C, left). This suggests that TgDAGT2 may have a nutrient dependent role, similar to

TgGPAT. The lack of an effect on parasite growth at higher lipid availability (1-10% FBS) could be explained through a compensation by its other homologue, TgDGAT1.

Previous studies were unable to generate a KO of this gene and the essentiality of this protein is still unclear (36).

To determine the possible compensating effect of these two DGATs, TgDGAT1 was knocked out in the TgDGAT2-mAID strain to generate a ∆TgDGAT1-TgDGAT2-HA- We wanted to also determine if the loss of both paralogues of DGAT in the parasite influenced parasite growth, and more over if this growth was affected by nutrient availability. We performed a plaque assay similar to the TgDGAT2-mAID strain (Fig. 3E). There was a general trend of increased parasite growth when both TgDGAT1 and TgDGAT2 were absent, but this effect was only significant in 0% FBS conditions (low lipid environment). This phenotype mirrors the TgDGAT2-mAID parasite strain, in that absence of TgDGAT2 (Fig. 3C) led to decreased parasite growth but disruption of both TgDGAT1 and TgDGAT2 led to an improvement of parasite development. This could suggest that at 0% FBS, these proteins are involved in controlling parasite growth.

TgGPAT is neutral lipid and phospholipid metabolism

To determine the nutrient-dependant roles of TgGPAT and TgDGAT2 in the lipid metabolism, we grew parasites in the presence or absence of auxin in 0-10% FBS to imitate nutrient (specifically lipids) starvation/excess and performed lipidomic analysis.

When the protein TgGPAT was downregulated, the parasites had a lower abundance of lipids at 0% to 1% FBS but at 10% FBS, the degree of decrease was not significant suggesting that the parasite can, somewhat, compensate for the lack of TgGPAT (Figure A). In the absence TgGPAT, there were many changes in the relative abundance of each FA species. In Figure 4B, the ratio of the abundance of each FA species with/without auxin (without/with TgGPAT) is plotted in a heatmap (where yellow is an increase and blue/purple is a decrease of abundance). We noticed that parasite lipid abundance of longer-chained FAs that are mostly host-derived such as C18:0, C20:0, C20:1, C20:4 and C20:5 is increased concomitantly to the increase of FBS (from 0% to 10% FBS). This suggests that with an increase in FBS levels, the Since there were no changes at 10% FBS in the presence of absence of the protein (and that the parasite can somewhat compensate their lipid content for the lack of TgGPAT at 10% FBS), we decided to focus our lipidomic analyses only at low lipid levels (0-1% FBS conditions, Figure A). We analysed the lipidomic changes the loss of this protein had on the proposed substrate FFA and other neutral/phospholipids.

There was a significant increase in FFA levels and a decrease in PL (comprising of LPA too, the proposed product of TgGPAT and simplest LPL) levels only at 1% FBS, considered to be low lipid availability (Figure C-D). The same changes were seen in 0% FBS but were not significant (Figure C).

Total TAG (0% FBS) levels were not affected, but the FA profile revealed that C14:0 (apicoplast-derived) decrease with an increase in stearic acid (C18:0), emphasizing the preference of this protein to longer chained FAs (

TgGPAT is involved in LPA formation

In these limiting lipid conditions, the loss of TgGPAT led to the decrease in the PL levels (Figure 4D and Sup. Fig. 4G/H) at 1% FBS. To investigate further we separated the total lipid extract by 2D-high performance thin layer chromatography (2D-HPTLC)

to allow us to determine the levels individual PLs and LPLs (Fig. 4J). There was a significant decline of PC levels, which is the majority PL group in most eukaryotes.

Importantly, we saw that LPA levels also decreased, which is in agreement with this LPL being the proposed product of TgGPAT (Figure J).

TgGPAT has a preference to using host-scavenged lipids

To determine if the parasites have any substrate preference, for FASII-derived FA or host-derived FA, metabolic labelling using isotope-labelled U-13 C-glucose were performed. To monitor the FASII-derived FA, the parasites were fed with U-13Cglucose, as previously described (31), under TgGPAT degradation by auxin treatment.

The incorporation rate of 13 C was determined for each FA species (Figure L). While FASII is fully active to produce the C14:0 fatty acid with fully 13 C labelled up M+14 (Fig. 4I), the abundance of FA generated from the FASII was much lower in the absence TgGPAT protein (Figure K). We also determine the abundance of FA scavenged from the host by growing the host cell in the presence of 13 C-glucose prior to the inoculating the parasite to grow with or without auxin, as we previously described (7). As shown in the Fig. 4 L, the parasite FA-scavenging abilities seemed to also be hindered (Figure L), specifically that of oleic acid (C18:1) and eicosenoic acid (C20:1) which is exclusively derived from the host. This is further supported by changes in the mass isotopologue distribution of host-derived C14:0, C16:0, C16:1, C18:0 and C18:1 (Fig. 4J-N, increase specifically in M+0 of C18:0). This suggests that the in the absence of TgGPAT, the parasite does in fact have a preference to host-scavenged lipids.

TgDGAT2 is involved in neutral lipid metabolism, specifically DAG metabolism Thereafter, to determine the role of TgDGAT2 in the lipid metabolism, we performed the same lipidomic analysis as for the TgGPAT-mAID. In contrast to TgGPAT, TgDGAT2 degradation led to an overall increase in total lipid content at all FBS conditions (only significant at 0 and 10% FBS, Figure 4A). There was no difference in the relative abundance of FA in the mol% (Sup. Fig. 5A).

Then the abundance of DAG and TAG, the proposed substrate and product for the TgDGAT2 was determined. There was an increase in the DAG at 0-10% FBS, specifically C16:0 (Figure B-D). However, the abundance of TAG was not affected.

To investigate the effect on TAG we preformed lipid droplet (LD) analyses (Figure E).

Lipid droplets were stained with nile red and its fluorescence was determined by IFA to quantify the number of LD based on iterative image processing (42). The partial loss of TgDGAT2 by auxin led to a significant decrease in the number of LD per parasites (0% and 1% FBS, Figure E) and volume (0% and 10% FBS, Figure D).

Since the FFA is also another putative substrate of TgDGAT2, its abundance was also investigated. As shown in the Fig. 5F and 5G, the relative abundance of C16:0 and C18:1 increased under 0% and 1% FBS when TgDGAT2 was disrupted. This suggests that a presence of this protein may give parasites a preference towards longer chained (possibly host-derived) FA species (Figure F/G). Since there is homologue TgDGAT1, it may compensate some part of the lipid metabolism thus the effect of the loss of TgDGAT2 was not seen lipidomically, but it was clearly shown that TgDGAT2 plays a role in LD formation.

Discussion

TgGPAT is a GPAT3-type AT that has a nutrient dependant role in neutral lipid and phospholipid synthesis with a preference of host FAs

We identified a GPAT3-type AT in T. gondii parasites by sequence analyses. The motif III of this protein has the highest similarity to the two yeast homologues (ScGPT2 and ScSCT1, Fig. 1 and Sup. Fig. 1A) rather than the human form, suggesting like with yeast that this protein may perform the roles of multiple GPAT-enzymes and that the parasite only houses two GPATs, TgATS1 (TGGT1_270910, plant/algal like) and

TgGPAT (31,49,50). Additionally, GPAT3 type proteins are unique in that they can perform the roles of AGPAT/GPAT and unlike GPAT1/2 it specifically localizes to the ER and not the mitochondria of the cell, as we have displayed by IFA colocalization studies with a Der1-GFP plasmid (Fig. 2B) (16). Since TgAGPAT is not essential when conditionally knocked out, it could be proposed that the existence of a plant-like AGPAT (TgATS2) and a dual-functioning TgGPAT could compensate for the loss of this AGPAT activity (16,26,32). This could also explain why when TgGPAT is knockeddown the parasite is not able to survive as potentially GPAT and AGPAT activity is inhibited (Fig. 2).

We were unable to show full alignment (only motif 3) of a TgGPAT 3D model with the crystal structure of CmATS1, which is like TgATS1 (31). Unlike plastid derived TgATS1 and CmATS1, TgGPAT is predicted to be poorly soluble, and there is currently no crystal structure of a membrane bound AT (40,46,51).

This work shows that TgGPAT crucial to parasite growth and is involved in neutral lipid and PL metabolism (Figure ). The absence of a significant drop in lipid content level when the protein is absent in lipid restraining conditions, can suggest that TgGPAT is more important under nutrient restrictions. In support of this hypothesis, changes in the individual neutral lipid class were only observed at 1% FBS, with a similar lipidomic phenotype at 0% but which was not significant (Figure C/D). This can be misleading, but when we look at the FA profiles of TAG and FFA, we see a trend; the loss of TgGPAT causes and accumulation of longer FA chains (C18:0 and C18:1), suggesting a preferred substrate-binding affinity to oleate acid and other longer chains like the human homology (22) and CmATS1 (40). This phenotype at low lipid conditions correlates a putative role in LD formation as LD predominantly form under low lipid levels (7,32). In other models, GPAT3 also possesses AGPAT activity which we did not investigate. It would be interesting to explore AGPAT activity using labelled LPA or enzymatic studies.

At 10% FBS (Fig. 4B), we see an exaggerated decline in C14:0 and an accumulation of FAs exclusively host-derived like eicosanoid acid (20:1) and arachidonic acid (20:4) (7). This is further supported through our metabolic host-labelled studies, which show a shutdown of parasite scavenging when TgGPAT is supressed. This accumulation of FA substrate leads to a 'lipotoxic' effect whereby the parasite believes it no longer requires FAs as they can no longer converted to PL, specifically the product LPA (Figure 4J), for membrane biogenesis. The effect this loss of LPA has on membrane integrity and curvature can be further investigated by electron microscopy as no visible membrane changes were noticed surveillance by phase-contrast microscopy.

These FAs are also not incorporated in neutral lipid synthesis (i.e. TAG/LD), as we see the loss of this protein leads to a suppression of LD formation at 1% FBS (Fig. 4E). Instead, these FAs accumulate in the parasite ultimately leading to cell death.

The unexpected shutdown of the FASII (de novo FAS), can be explained by this fatal accumulation of FAs in the parasite (Figure L).

TgDGAT2 has a nutrient dependant role in TAG formation

Phylogenetic and evolutionary analyses show that the DGAT1 and DGAT2 families have asymmetrically evolved since the rise of eukaryotes and have undergone convergent evolution likely due to the importance of TAGs for all eukaryotic organisms (25). A DGAT1 isoform exists in the parasite (TGGT1_232730) and the sequence motif analyses performed in this study, clearly indicated that TgDGAT2 is not part of the same superfamily of ATs (36). In P. falciparum parasites previous studies show the existence of a single DGAT1 (no DGAT2), which could be detected by antibodies against TgDGAT1, suggesting that PfDGAT is the most likely the only candidate for plasmodial triacylglycerol synthesis in this parasite unlike Toxoplasma (47,52,53).

Following structural and sequence analyses of TgDGAT2, it was surprising to find that despite having 2 transmembrane domains this protein seemed to localize evenly throughout the parasite (Figure B). Under auxin treatment, IFA revealed we were unable to completely knockdown the protein, which instead seemed to accumulate in the apicoplast. We suspect this is a default using the mAID system, even if auxin can cross all 4 membranes of the apicoplast, perhaps the recruitment of the SCF TIR1 complex to the ubiquitinated protein in the apicoplast is not possible (48), resulting in a failure in protein degradation. Alternatively, this could be a background signal of the apicoplast since western blot analyses revealed complete protein loss (Figure A). The IFA analyses of the double mutant line (Tg∆DGAT1-DGAT2-iKD, Fig. 3D) further supported the predicted membrane-bound nature of TgDGAT2, as it has a perinuclear/ER cellular localization like the DGAT1 isoform (36).

Tables Table 1: A C-terminally tagged HA line with inducible post-translational protein degradation was generated using the Auxin-inducible Degron system (AID). Homology regions of the C-terminal end of TgGPAT/TgDGAT2 (not shown here) excluding the stop codon were amplified using the primers found in Tables Table 1. PCR (Table 1) were preformed using mutant parasite DNA to show correct integration of the mAID sequence, the HX drug cassette and correct gene insertion.

2013; [START_REF] Ludewig-Klingner | Distribution and Evolution of Peroxisomes in Alveolates (Apicomplexa, Dinoflagellates, Ciliates)[END_REF]Moog, Przyborski and Maier, 2017). This protein is peroxisomal too and activates FAs destined for energy generation via β-oxidation, which instead of the mitochondria could possibly exists in a peroxisomal-like organelle.

The close association of peroxisomes and LDs in other eukaryotic model, emphasize that this could also occur in the parasite (Kohlwein, Veenhuis and van der Klei, 2013).

Additionally, I show that this process is most probably only induced under certain circumstances such as extracellular conditions or when nutrients are scarce. It is crucial to identify the metabolites of the β-oxidation pathway to understand when, why and even if this process occurs in Apicomplexa.

The protein needed for the correct localization of FAs into the mitochondria to allow for FA oxidation (CPT1a) is absent in Toxoplasma and Plasmodium, hence why peroxisomal FA oxidation seems more probable [START_REF] Danne | Alveolate Mitochondrial Metabolic Evolution: Dinoflagellates Force Reassessment of the Role of Parasitism as a Driver of Change in Apicomplexans[END_REF]. The lack of peroxisomal import proteins and enzymes in plasmodial parasites suggest that the occurrence of conventional peroxisomes is unlikely (Mcintosh, Elliott and Joiner, 2005).

Some proteins involved in the biogenesis of peroxisomes (PEX proteins) also exist in Toxoplasma and P. falciparum parasites. To date, there is still no microscopic evidence of this organelles and we do not know how they are involved in metabolic processes [START_REF] Ludewig-Klingner | Distribution and Evolution of Peroxisomes in Alveolates (Apicomplexa, Dinoflagellates, Ciliates)[END_REF]Moog, Przyborski and Maier, 2017). Additionally, the nutrient conditions of previous work (which were predominantly bioinformatical) were often overlooked and it is apparent that the lipid/nutrient availability can drastically change the metabolic choices made by the parasite (Shunmugam et al., 2021;Shunmugam, Arnold, et al., 2022;Walsh et al., 2022).

Future work should entail deciphering the metabolome of these parasites under conditions known to induce β-oxidation such as excess FAs or glucose/amino acid starvation. Electron microscopy would also be necessary to identify peroxisomal like vesicles and what other organelles they associate with (i.e. lipid droplets). Alternatively, the metabolome/lipidome of the parasite's unique mitochondrion should be characterized. The loss and repurposing of certain proteins could suggest that a βoxidation process that is different from the host, can potentially be targeted through therapeutic strategies.

Lipid mechanisms of sexual conversion:

The discovery that LPC regulates sexual stage conversion in P. falciparum parasite was a turning point in the field Apicomplexa biology (Brancucci et al., 2017). This work stressed how important the balance of PL and lyso-PL is in parasite development and disease propagation. Previous work has also highlighted this by characterizing a phospholipase, an enzyme responsible for the hydrolysis of a FA from the phosphorglycerol backbone of PC resulting in LPC [START_REF] Singh | Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission[END_REF][START_REF] Flammersfeld | A patatin-like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum[END_REF]. This specific group of phospholipases A2 enzymes (PLA2) are known to mediate membrane remodelling and vesicle secretion in organisms. Numerous phospholipases exist in Plasmodium which may or may not have redundant function.

My thesis focused on the investigation of a phospholipase that is highly expressed in schizonts and sexual stage parasites and is critical when lipid deprived.

Lipidomic analyses show that PfPNPLA2 is responsible formation of LBPA from PG.

LBPA is a lipid normally localized to the plasma membrane, hence this protein's importance for membrane biogenesis and parasite division. LBPA (BMP) and PG seems to have an indirect relationship. Previous work show that PG levels are lowered and LBPA levels are heightened upon sexual commitment in red blood stages (Gulati et al., 2015;[START_REF] Tewari | Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite[END_REF]. My thesis work only focussed on asexual stages.

Future perspectives would include viability and metabolic studies on sexual stages to determine if indeed this protein plays a role in sexual conversion. Heterologous complementation and protein kinetics could be used to confirm enzyme function.

Additionally, the role of this protein in nutrient deprivation must be further studied as the limitation on parasite survival in these conditions resulted in poor sample yield for lipidomic analyses. It would be important to understand the lipidic mechanisms of sexual conversion, since targeting this process can ultimately hinder disease transmission.

How ATs play an indispensable role in parasite lipid droplet biogenesis and lipid detoxification:

Investigating the role of two acyltransferases in Toxoplasma parasites was the final part of my thesis work. Both TgGPAT and TgDGAT2 have not been previously characterized and the results gathered here are novel to the field. These proteins have a strongly nutrient dependant role in the formation of either LPA or TAG, both crucial products of the glycerolipid pathway which is essential for membrane biogenesis and lipid detoxification (LD formation).

Unlike their hosts, Toxoplasma parasites have evolved to contain 2 sets of ATs (GPAT, AGPAT and DGAT) involved in the either an ER or apicoplast-based glycerolipid process. TgGPAT like its plastid paralogue, TgATS1 is essential under lipid starvation and enrichment (Amiar et al., 2016). This protein is essential to parasite development and its loss results in deleterious effects due to an accumulation of FAs followed by a lipotoxic death.

The sequential ATs, TgAGPAT and TgATS2 (plastid-origin), are not similar since their absence have no significant effects on parasite survival. Therefore, I hypothesize that

TgGPAT, which we now know is a GPAT3-type AT, may act as both a GPAT and AGPAT [START_REF] Sukumaran | Functional characterization of the human 1acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3[END_REF]. The exact enzymatic mechanisms of this protein should be investigated by complementation studies or enzymatic assays.

Throughout my thesis works, there is lipid group that is consistently affected, TAGs.

This lipid group is the major component of LDs and seems to be crucial in the detoxification of lipids and prevents parasite death (Shunmugam et al., 2021). Hostlipid droplet biology is deeply studied (considering they are larger and more numerous) in these parasites than their own LD biogenesis mechanisms [START_REF] Nishikawa | Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma[END_REF][START_REF] Romano | Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole[END_REF]Nolan, Romano and Coppens, 2017b;Nolan et al., 2018;[START_REF] Pernas | Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma gondii by Limiting Its Uptake of Fatty Acids[END_REF][START_REF] Vallochi | Lipid Droplet, a Key Player in Host-Parasite Interactions[END_REF]. Our lab is starting to unravel certain concepts about parasite LD biology, and this can be achieved by identifying and characterizing putative LD association proteins and other ATs.

Solubility issues with TgGPAT, a membrane bound AT, may pose certain limitations when attempting to isolate the protein (Shabits, 2017). Instead, complementation assays using an optimized codon sequence in a hybrid yeast mutant may be a solution.

This protein is extremely important to parasite survival, and it would be worth investigating its druggability.

TgDGAT2, on the other hand seems to only have two-transmembrane domains and may be more soluble that TgGPAT. This protein and its paralogue DGAT1, are essential in LD biogenesis (in other eukaryotic models) and are usually in association with other proteins like SEIPIN, MOSPD, VAP-B etc., but binding partners/interaction are completely unknown in these parasites [START_REF] Liang | Glycerolipids DGAT enzymes and triacylglycerol biosynthesis[END_REF][START_REF] Zhang | Lipin proteins and glycerolipid metabolism: Roles at the ER membrane and beyond[END_REF]Bhatt-Wessel et al., 2018b;[START_REF] Choudhary | Architecture of Lipid Droplets in Endoplasmic Reticulum Is Determined by Phospholipid Intrinsic Curvature[END_REF][START_REF] Combot | Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes Graphical abstract[END_REF].

My PhD work showed the protein essentiality in this parasite is highly dynamic and strongly dependant on nutrient availability. I show how this capability can influence metabolic reprogramming (i.e. inducing β-oxidation), parasite stage conversion and lipid detoxification (LD vs membrane biogenesis). Collectively this brings novel data on how plastic and metabolically different theses parasites are from their hosts. This work contributes to developing novel therapeutics in combating these diseases.

  FIGURES ............................................................................................................

  ............................. 315 Chapter 7: Conclusion and perspectives ........................................................... 320 Deciphering the putative peroxisomal metabolome in Apicomplexa: .................. 321 Lipid mechanisms of sexual conversion: ............................................................. 323 How ATs play an indispensable role in parasite lipid droplet biogenesis and lipid detoxification: ...................................................................................................... 324 REFERENCES ................................................................................................... 327

Figure 1 : 35 Figure 2 : 37 Figure 3 : 40 Figure 4 :

 1352373404 Figure 1: Life cycle of the malaria parasite, P. falciparum. ....................................... 35

Figure 5 :

 5 Figure 5: Lipid pathways of the metabolic organelles found in Apicomplexa parasites.

Figure 6 :

 6 Figure 6: Metabolic pathways in the apicoplast of apicomplexan parasites. .............

Figure 7 :

 7 Figure 7: The metabolic pathways of the mitochondrion of apicomplexa parasites. .

Figure 8 :

 8 Figure 8: A schematic representation of phospholipid synthesis pathways in T. gondii

Figure 9 :

 9 Figure 9: The levels of phosphatidylglycerol, lysobisphosphatidic acid and

Figure 1 :

 1 Figure 1: Life cycle of the malaria parasite, P. falciparum.

Figure 2 :

 2 Figure 2: Lifecycle of Toxoplasma gondii parasites.The definitive host of T. gondii parasite are felids, where sexual development occurs. The oral ingestion of a bradyzoite cyst equally leads to its rupture once entering the intestinal tract of the host. Individual trophozoites from this cyst will enter epithelial cells. The parasite then enters the first round of schizogony (asexual) that results in the development of schizonts which will lyse the intestinal cell that will commence gamogony through the development of female and male gametes. The fusion of these two cells will lead to the formation of and oocyst that is eventually excreted out of the felid as sporulated oocysts in the environment that can last months to years. The ingestion by other mammals or birds will then release the sporozoites of the sporulated oocysts in the intestinal tract of the intermediate host. Asexual development will occur first in the epithelia cells that will release hundreds of tachyzoites that can invade any nucleated cell. Monocyte invasion will facilitate the migration of the tachyzoite parasite to the eye, brain, placental or heart tissue where bradyzoite conversion will occur. Cysts that form

Figure 3 :

 3 Figure 3: Structure of Apicomplexa parasites.

falciparum and 8

 8 or more in T. gondii); club shaped structures which are rich in proteins and lipids, b) micronemes; rod-shaped electron dense structures and c) dense granules; which are all central to parasite invasion and proliferation in its host. The apical organelles are enclosed by an intricate structure consisting of a) the conoid which is an assembly of spirally arranged fibres, b) the polar ring, c) two intraconoidal microtubules, and the d) the preconoidal rings. The structures described here are visibly apparent during the merozoite and tachyzoite stages in P. falciparum and T.

Figure 4 :

 4 Figure 4: Schematic representation of active invasion process for T. gondii and P. falciparum parasites. The invasion process (from left to right) of apicomplexa parasites consists of parasite attachment, reorientation/junction formation, parasitophorous vacuole formation and mature invasion. The proteins involved for each process for T. gondii tachyzoites (top) and P. falciparum merozoites (bottom) are indicated in red. GPI-MSP: glycosylphosphatidylinositolmerozoite surface proteins, MIC2: microneme protein 2, AMA1: apical merozoite antigen 1, RON: rhoptry neck proteins, ROP: rhoptry bulbous body proteins, Rh: rhoptry proteins, GRA: dense granule proteins, SUB: subtilisin-like serine proteases.

Figure 5 :

 5 Figure 5: Lipid pathways of the metabolic organelles found in Apicomplexa parasites.The lipid metabolic pathways can be found in the ER (endoplasmic reticulum), the apicoplast, the mitochondrion, the peroxisome, and the lipid droplets (LD) of the parasite. Phosphoenolpyruvate (PEP) and DHAP (dihydroxyacetone phosphate) generated from glycolysis occurring in the cytosol of the parasites enter the apicoplast via the apicoplast phosphate transporter (APT). They are then converted to pyruvate and glycerol-3-phospahte (G3P) by a PK (pyruvate kinase) and GpdA (glycerol-3-phosphate dehydrogenase), respectively. A PDHC (pyruvate dehydrogenase complex, only present in the apicoplast) is responsible for the formation of acetyl-co-enzyme A (CoA) from pyruvate. Acetyl-CoA carboxylase (ACC) converts this to malonyl-CoA. FabD/H complex leads to the attachment of an acyl carrier protein (ACP) and the loss of CoA resulting in ACP-malonyl. The fatty acid synthase II (FASII) complex (comprised of FabG/ B/F/I/Z) are responsible for the formation of

Figure 6 :

 6 Figure 6: Metabolic pathways in the apicoplast of apicomplexan parasites. Four metabolic processes exist within the apicoplast, isoprenoid (IPP) synthesis, fatty acid (FA) synthesis, Heme synthesis and iron-sulphur (Fe-S). DOXP: 1-deoxy-D-xylulose 5-phosphate, MEP: mevalonate/methylerythritol phosphate (MEP), NADH: nicotinamide adenine dinucleotide, ACP: acyl carrier protein, CoA: co-enzyme A. Adapted from(Ralph et al., 2004)).

Fatty

  acids (FA) in other eukaryotic models can contribute to acetyl-CoA production via βoxidation in the mitochondria or peroxisomes which is hypothesized to be similar for apicomplexan parasites. A carnitine palmitoyl transferase I (CPT) to allow for the transport of FA (namely C16:0) into the matrix does not exists in T. gondii and P. falciparum parasites.

Figure 7 :

 7 Figure 7: The metabolic pathways of the mitochondrion of apicomplexa parasites.

Figure 8 :

 8 Figure 8: A schematic representation of phospholipid synthesis pathways in T. gondii and P. falciparum parasites. Enzymes (in brown circles) are indicated here that allow for the formation of major phospholipid (PL) groups, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylthreonine (PT). Choline (Cho-orange circle), lysophosphatidylcholine (LPC), serine (Ser-pink circle) and ethanolamine (Etn-blue circle) can be important from the host cell into the parasite. Serine can also be derived from the degradation of heme/haemoglobin in malaria parasites. Enzymes unique to either parasite are indicated with either a pink plasmodial merozoite or green Toxoplasma tachyzoite. Enzymes that are common in other eukaryotic models and yet to be characterized in these parasites are indicated by a grey square. Choline kinase: CK, choline phosphate: cytidyltransferase: CCT; diacylglycerol choline phosphotransferase: CPT, phosphatidylthreonine synthase: PTS, phosphoethanolamine methyltransferase: PMT; phosphatidylethanolamine Nmethyltransferase: PEMT, serine decarboxylase: SD, ethanolamine kinase: EK, ethanolamine phosphate: cytidyltransferase: ECT, diacylglycerol ethanolamine phosphotransferase: EPT, phosphatidylserine synthase: PSS, phosphatidylserine decarboxylase: PSD, phospholipase A: PLA (hydrolysis of fatty acid on sn-1/2 position) phospholipase C: PLC (hydrolysis of phosphate group), phospholipase D: PLD (hydrolysis of R-group on phosphate head),

Figure 9 :

 9 Figure 9: The levels of phosphatidylglycerol, lysobisphosphatidic acid and lysophosphatidylglycerol in RBC stage P. falciparum parasites. Phosphatidylglycerol (PG) levels decrease as parasites transition to sexual development. The levels of lysobisphosphatidic acid (LBPA) and lysophosphatidylglycerol (LPG) seem to concomitantly increase with this conversion. It is hypothesized that a stage-dependant

  and this highlight how the parasite can adapt to different nutrients conditions to escape lipotoxic-induced death by channelling to TAG production for LD and preventing uncontrolled synthesis of membranes (see Chapter 3: A crucial lipid equilibrium governs the survival of asexual Apicomplexan parasites). During my thesis I was the first author of a review entitled: 'The flexibility of Apicomplexa parasites in lipid metabolism', in which the role/pathways of lipids in Apicomplexa parasites are discussed along with a summary of recent findings on the metabolic mechanisms in host nutrient adaptation (Shunmugam et al., 2022, see later in this chapter).The work discusses the following. Summary of published review (Shunmugam, Arnold, et al., 2022): • Lipids are essential to parasites survival, especially for the stages that are rapidly dividing like blood stage P. falciparum parasites and T. gondii tachyzoites. • These parasites conform to a widely accepted lipid metabolism dogma: o 'MAKE' fatty acids through the apicoplast FASII pathway o 'TAKE' fatty acids directly from the host cell or the host environment o Combine these fatty acids into the different lipid classes contributing to membrane biogenesis, lipid signalling and lipid storage • Scavenging of different lipids that are not made by the parasite are different in Toxoplasma and Plasmodium parasites (also depending on the life stage) and the mechanisms are described in this review.

o

  Ces parasites se conforment à des mécanismes métabolique communément accepté : Synthétiser des acides gras via la voie FASII dans l'apicoplast o Consommer des acides gras prélevés directement dans la cellule hôte ou son environnement o Combiner ces acides gras pour synthétiser les différentes classes de lipides essentielles à la biosynthèse membranaire, la signalisation lipidique et le stockage.

  an essential phosphatidic acid phosphatase (TgLIPIN), which acts as a regulator for this equilibrium between lipid synthesis and storage. The major contributions of this study include: • TgLIPIN (the only LIPIN encoded in the T. gondii genome) is an essential protein for parasite survival and has active phosphatidic phosphatase activity allowing for the conversion of PA to DAG • Down-regulation of this protein induces rapid membrane malformation, cytokinesis and replication defects which is aggravated in a high lipid environment o This predominantly induces excessive biogenesis of nuclear/ER membranes and the IMC • This protein prevents lipotoxic parasite death in a high lipid host environment by channelling the flux of host fatty acids and prevents the accumulation of FFA and promotes TAG synthesis in the form of LD My contribution to the work included: • Heterologous yeast complementation studies proving the phosphatase activity of this protein • lipidomic and lipid flux analyses • Illustrations and writing of the paper Résumé de l'article publié (Shunmugam, Dass et al., 2021): Le travail présenté dans ce papier pose les fondations de ma thèse et a été initié par Dr. Sheena Dass. Le métabolisme lipidique des parasites apicomplexes demande de combiner des acides gras provenant de différentes sources pour former les fragments de lipides décrits précédemment. Les flux d'acides gras d'origines multiples sont gouvernés par la disponibilité des nutriments trouvé dans la cellule hôte et son environnement. Un équilibre précis doit être maintenu entre la synthèse de lipides et leur stockage pour permettre au parasite de se développer efficacement. Notre travail a permis la caractérisation d'une phosphatase d'acide phosphatidique (TgLIPIN), qui agit comme un régulateur de cet équilibre.

2 .

 2 Assess the essentiality, localization, and cellular context of each target protein in both models 3. Determine the function of the target protein at the parasite lipid synthesis level using core lipidomics by GC-MS, and lipid flux analyses by stable isotope labelling combined to lipidomics 4. To understand the global contribution of each protein class to the parasite's nutrient-adaptability in lipid synthesis, storage, and utilization processes CHAPTER 4: PUTATIVE TOXOPLASMA GONDII ACYL-COA SYNTHETASES ARE INVOLVED IN NEUTRAL LIPID METABOLISM UNDER VARIED NUTRIENT CONDITIONS A putative peroxisomal Toxoplasma gondii acyl-CoA synthetase (TgACS1) is involved in neutral lipid metabolism under suboptimal nutrient conditions Lipid biosynthesis in parasites, as with all other organisms is reliant on the metabolic activation of fatty acids (FAs) by the parasite. Acyl CoA synthases (ACS) catalyse the thioesterification of FA, allowing their metabolic use by any enzymes handling FAs in the parasite. Beside the crucial functions of FA for parasite survival, the presence and roles of ACS is yet to be determined. In P. falciparum and C. parvum 13 and 3 ACS proteins have been defined, respectively. We identified 7 putative ACSs encoded by the genome of T. gondii (TgACS). The differential localization of these proteins suggests that ACS in this parasite have varied functions. We prioritized a protein which seemed to have an important role in parasite survival when lipid levels are scarce in the host, hence suggesting a function in parasite metabolic adaptation; TgACS1. The major findings of this are listed below: • TgACS1 is important in FFA formation and TAG utilization in intracellular parasites in low-nutrient conditions for the host. • This is also true for extracellular parasites, where the phenotype of TgACS1 disruption is even more marked than in intracellular parasites • TgACS1 is important for the utilization of LD in extracellular parasites and its disruption affects parasite motility/energy production • TgACS may be important for directing FAs toward the β-oxidation pathways under low nutrient levels • Heterologous complementation in yeasts conclude that TgACS1 has peroxisomal acyl-CoA synthétase function Résumé du manuscrit : Une acyl-CoA synthétase (TgACS1) péroxisomale putative chez Toxoplasma gondii est impliquée dans le métabolisme des lipides neutres en conditions nutritives défavorables. La biosynthèse de lipides chez les parasites, comme pour les autres organismes, requiert l'activation préalable des acides gras. Les acyl-CoA synthétase (ACS) catalysent la thioestérification des acides gras libres, permettant leur utilisation dans le métabolisme par toutes les enzymes dont ils sont le substrat. Outre le rôle crucial des acides gras pour la survie des parasites, la présence et fonctions des ACS restent à déterminer. Treize ACS ont été identifiées chez P. falciparum et 3 ACS chez C. parvum. Nous avons identifié 7 ACSs putatives dans le génome de T. gondii (TgACS). Les différences de localisation de ces protéines suggèrent que les ACS de ce dernier occupent des fonctions variées. Nous avons priorisé la protéine TgACS1 dont le score d'essentialité évalué était élevé et pour laquelle la déplétion avait le plus fort impact sur la viabilité du parasite en conditions de stress nutritif, suggérant ainsi une fonction dans l'adaptation métabolique du parasite Les résultats principaux sont les suivants : • TgACS1 est importante pour la formation des acides gras libres et pour la consommation des triglycérols chez le parasite intracellulaire en conditions de stress nutritif. • Cette observation se vérifie également chez les parasites extracellulaires pour lesquels la disruption de TgACS1 induit un phénotype encore plus sévère que pour les parasites intracellulaires. • TgACS1 permet la consommation des gouttelettes lipidiques chez les parasites extracellulaires et sa disruption diminue la mobilité, et donc production d'énergie, du parasite • TgACS pourrait contribuer à la consommation d'acides gras via les voies de βoxydation en conditions de stress nutritif. • Des expériences de complémentation hétérologue conduites chez les levures confirment que TgACS1 possède une fonction d'acyl-CoA synthétase peroxysomale. A putative peroxisomal Toxoplasma gondii acyl-CoA synthetase (TgACS1) is involved in neutral lipid metabolism under sub-optimal nutrient conditions Serena Shunmugam 1 , Sheena Dass 1 , Christophe-Sébastien Arnold 1 , Sarah Charital 1 , Samuel Duley 1 , Fabien Pierrel 2 , Yoshiki Yamaryo-Botté 1 *, Cyrille Y. Botté 1 * 1. Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Université Grenoble Alpes, INSERM U1209, Grenoble, France, 2. Université Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, 38000Grenoble, France.

  diseases caused by apicomplexans are responsible for major social and economic burdens on more than 1/3 of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments, all argue for the urgent need to develop new therapeutical tools to combat these diseases. Understanding the key metabolic pathways sustaining hostintracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current data support parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unravelled. Our results clearly indicate the role of acyl-CoA synthetases for the essential metabolic activation of FA used to maintain parasite propagation and survival. The significance of our research is 1) the identification and cellular localization of 7 of these enzymes in T. gondii parasites, 2) using lipidomic techniques we also identified a nutrient dependent role of TgACS1, 3) yeast complementation assay showed that ACS1 has acyl-activating function and possible implication in the βoxidation 4) and has a important role for extracellular parasite motility and energy production.INTRODUCTIONApicomplexa parasites are a group of unicellular eukaryotes that include obligate intracellular parasites responsible for major human and cattle diseases. These include Toxoplasma gondii, Plasmodium sp. and Cryptosporidium parvum, which are the causing agents of the infectious diseases toxoplasmosis, malaria and cryptosporidiosis, respectively. Eradicating these diseases is a worldwide priority, and the renewal of our therapeutic arsenal relies on understanding host-parasite interactions sustaining parasite survival. Specific metabolic pathways allowing nutrient acquisition, intracellular development, and parasite propagation represent ideal targets for drug development. Lipid synthesis, acquisition and homeostasis have been shown

  imported into the peroxisomal lumen bear two canonical targeting sequencesperoxisomal 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[RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X-[HQ]-[LAF](21). 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(39). This peroxisomal LACS activity has been shown to be essential for seedling growth(39).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(21). TgACS1 was one of the proteins defined within a "high confidence" PTS1-targeted peroxisomal proteome for T. gondii. Vesicular localization and predicted PTS1 at the C-terminus of TgACS1 further strengthen its putative role in the elusive βoxidation pathway in T. gondii. Co-localization studies with peroxisomal markers like catalase can provide further confirmation. Electron microscopy of extracellular parasites under nutrient-stress conditions would be able to confirm the existence of peroxisomal-like vesicles, previously not known.

  Phylogeny: Identification of TgACSsMembers of the Acyl CoA synthetase (ACS) gene family within T. gondii were //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.

(

  NX_Q5FVE4); Chromera velia (Cvel_26948), Chromera velia (Cvel_29920), C. velia (Cve1_20092), C. velia (Cvel_8845)), C. velia (Cvel_24841), C. velia (Cvel_29002), C. velia (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.

  7 g/l of Yeast Nitrogen Base (YNB, MP biomedicals), 0.77 g/l of CSM-URA (MP Biomedicals) and g/l of glucose, as described. In complementation experiments, the media were supplemented with 22.5 mM cerulenin (Sigma-Aldrich), 100 mM FFA, and 0.2% (v/v) final concentration. The different FFAs were dissolved in 100% TWEEN 20 at a concentration of 50 mM. Cerulenin was dissolved in acetone at 45 mM. As controls, media without FFAs and/or cerulenin were also supplemented with 0.2% (v/v) TWEEN 20 and/or acetone.Three clones per transformation were used for complementation tests. For each experiment, one colony was inoculated in 6 mL of CSM-URA + glucose (2%) and grown overnight at 30⁰C at 180 rpm. Growth assays were performed in 96-well microplates (Thermo Scientific Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate) filled with 200 mL of culture in glucose medium supplemented or not by FA or cerulenin and set at an OD600 =0.1. The microplate was incubated at 30⁰C in an Infinite M2000 PRO microplate reader (Tecan) set for plate orbital shake for 10 s and absorbance measurements (OD600) every 20 min, with four measurements per well. OD600 readings from experimental wells minus the original OD600 values resulted in the ∆OD600 values shown in Sup. Fig.5.

FIGURESFigure 1 :

 1 FIGURES

Figure 2 :Figure 3 :

 23 Figure 2: TgACS1 is a protein containing a bubble-gum domain and PTS1 signal

Figure 4 :

 4 Figure 4: TgACS1-iKD is important for extracellular parasite motility and lipid

Figure 5 :

 5 Figure 5: Growth complementation of a yeast mutant deficient of peroxisomal acyl-CoA activity by TgACS1. In the absence of doxorubicin, the ∆PCS60 strain

Figure 6 :

 6 Figure 6: Model explaining the proposed role of TgACS1 in parasite host-

  TgACS3, via a Tet-off-based inducible KD parasite line, revealed that the protein is critical for tachyzoite intracellular development. Cellular characterization shows that TgACS3 is required to maintain intracellular propagation of T. gondii tachyzoites, especially under high host nutrient environments. Lipidomic and fluxomics analyses further determined that the disruption of TgACS3 significantly reduces the parasite lipid content, phospholipid content, and concomitantly accumulates host FA within the parasite. Together, our data reveal that TgACS3 is responsible for channeling the constant flux of host FA to generate the bulk of phospholipid for parasite membrane biogenesis, hence maintaining tachyzoite division and survival.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Cellular characterization of TgACS3-iKD reveal its critical role for

Figure 3 :

 3 Figure 3: Lipidomic analyses of TgACS3-iKD mutant under high and low host

Figure 4 :

 4 Figure 4: Lipid flux analyses of host-derived lipids in the TgACS3-iKD mutant under high host nutrient content. Using 13 C stable isotope-labelled host cells, lipid

Figure 5 :•••••

 5 Figure 5: Model explaining the proposed roles of TgACSs in parasite host-

  ABBREVIATIONS

  )phosphate). In eukaryotes, LBPA is a lipid class normally involved in slow catabolism, such as degradation of lipid stores for energy production in nutrient poor conditions[START_REF] Goursot | Structure, Dynamics, and Energetics of Lysobisphosphatidic Acid (LBPA) Isomers[END_REF]. LBPA is negatively charged at the acidic pH of lysosomes, and these charges are central to its role in the degradation of lipids and membranes in the lysosome. In effect, these negative charges facilitate the adhesion of the soluble positively charged hydrolases and activator proteins (such as PLAs), thus allowing them to degrade the lipids at the interface of the inner membranes of the lysosome[START_REF] Gallala | Biological function of the cellular lipid bmp-bmp as a key activator for cholesterol sorting and membrane digestion[END_REF]. Here, our results indicate that PfPNPLA2 acts on the mitochondrial degradation of PG to form LBPA. Whether the synthesis of LBPA is important for blood stage parasitesurvival is an open question.

Figure 1 :

 1 Figure 1: PfPNPLA2 localizes to the apicoplast and mitochondria during

Figure 2 :

 2 Figure 2: PfPNPLA2 is important for blood stage replication under limiting lipid

Figure 3 :

 3 Figure 3: PfPNPLA2 depletion influences the lipidomic profile of asexual stage

Figure 4 :

 4 Figure

Figure 5 :

 5 Figure 5: PfPNPLA2 is involved parasite development via the conversion of PG

  1,250 bp for the PfPNPLA2-HA-KD; 1,075 for the PfPNPLA4-HA-KD). (C) Western blot analyses of rings, trophozoites (TR), schizonts (SZ), immature gametocytes (imGC, stage I-IV), mature gametocytes (mGC) and activated gametocytes (aGC, 30 min p. a.) of the (untreated) PNPLA2-KD using mouse anti-PNPLA2, anti-HA and anti-Pf39.

  acid (PA), diacylglycerol (DAG) and triacylglycerol (TAG). LPA and PA are the simplest PLs/PLs precursors and are crucial for a) cell signalling to induce parasite invasion, motility, and egress and production of DAG (via DGK1/2) (9,10), b) membrane curvaton for cytokinesis and endocytosis (11), and c) are considered the central precursors for all PL like phosphatidylcholine (PC), phosphatidylethanolamine (PE)

  cerevisiae SCT1 (P32784), Trypanosoma brucei GPAT (Tbg972.10.3920), Eimeria falciformis GPAT (ASN64451.1) Plasmodium falciparum GAT (PF3D7_1212500), Neospora caninum GPAT (NCLIV_029980), Hammondia hammondi GPAT (HHA_256980, Homo sapiens DGAT2 (Q96PD7), Arabidopsis thaliana DGAT2 (Q9ASU1), Saccharomyces cerevisiae DGAT2 (Q08650), Plasmodium falciparum DGAT (PF3D7_0322300), Neospora caninum DGAT2

Figure 1B .

 1B Figure 1B. This protein is predicted to be 533 aa and contains only 2 transmembrane

  mAID line. The TgDGAT1 gene was replaced with a GFP sequence, and IFA confirmed a disruption of the gene by a nuclear localization of the GFP signal. IFA analyses of ∆TgDGAT1-TgDGAT2-HA-mAID revealed that in contrast to TgDGAT2-mAID, TgDGAT2 has a perinuclear/ER like localization (Figure D).

  absence of TgGPAT leads to an accumulation of longer chained FA (possibly hostderived; Figure B and Sup. Fig 4A-C).

  Figure E). A similar change was seen for FFA levels at 0% FBS (Figure F), TAG (Figure G) and DAG at 1% FBS (Figure H). The protein's role in neutral lipid synthesis, specifically TAG/LD was highlighted with the decrease of LD volume at 1% FBS (Sup. Fig 4F).

FiguresFigure 1 : 1 )

 11 Figures

Figure 2 :

 2 Figure 2: Cellular phenotypic characterisation of TgGPAT.A) Western blot shows TgGPAT downregulation in TgGPAT-mAID iKD parasite line, in less than 30min after adding 100µM Auxin. TgGPAT (120 kDa) was probed with anti-HA antibody and TOM40 (40 kDa) was used as the loading control. B) IFA showing TgGPAT (in green) in close vicinity of ER marker Der-1 (in red). C) Plaque assays performed to determine changes in parasite growth after depletion of TgGPAT, showed significant effects in the presence of 0%, 1% and 10% FBS. The plaque area percentage (right) of TgGPAT-mAID iKD (+Auxin) declined by 4-5% in 0% and 1% FBS and even more in 10% FBS, depicting a strong growth defect. There is a statistical significance between the treated cultures in conditions of 0% vs 1% FBS and 0% vs 10% FBS, suggesting a 'rescue effect' in a high lipid-nutrient environment. D) A replication assay revealed significant difference between the relative abundance of each number of parasites of vacuoles between treated and untreated TgGPAT-mAID iKD cultures, depicting a growth defect in the absence of the protein. Scale bars= 2 µM. 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 indicated in each graph.

Figure 3 :

 3 Figure 3: Cellular phenotypic characterization of TgDGAT2 A) Western blot shows TgDGAT2 downregulation in TgDGAT2-mAID iKD parasite line, after of 100 µM auxin treatment. TgGPAT (120 kDa) was probed with anti-HA antibody and TOM40 antibody (40 kDa) was used as the loading control. B) IFA showing down regulation of TgDGAT2 (in green) under the same conditions. Even with auxin treatment there is an accumulation of immunofluorescence signal in the apicoplast (anti-CPN60 in red). C) Plaque assays performed to determine changes in parasite growth after depletion of TgDGAT2 under 0%, 1% and 10% FBS. The plaque area percentage (right) of TgDGAT2-mAID iKD (+Auxin) declined by 5% in 0% FBS, depicting a strong growth defect under nutrient stress (a.u.-arbitrary units). D) IFA showing down regulation of Tg∆DGAT1-DGAT2-iKD, with TgDGAT2 being perinuclear and the ∆TgDGAT1-GFP signal in the nucleus as expected. Scale bars= 2 µM. 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 indicated in each graph.

Figure 4 :

 4 Figure 4: Lipidomic analyses of TgGPAT-mAID-iKDA) A ratio of the total lipid content without/with TgGPAT in parasites under 0-10% FBS conditions, p-values are compared to 1 (unchanged levels). B) A heatmap displaying a ratio of fatty acid (FA) abundance in nmol/parasite(10 8 ) without/with TgGPAT in parasites under 0-10% FBS, yellow and purple indicate a fold increase or decrease in FA species, respectively. Mol% of each FA species found in triacylglycerol (TAG), free fatty acids (FFA), diacylglycerol (DAG) and phospholipids (PL) found in the total lipid content of parasites in C) 0% and D) 1% FBS with (grey, -auxin) and without (red, +auxin) TgGPAT. Mol% of FA species of E) TAG and F) FFA levels at 0% FBS with (grey) and without (red) TgGPAT. Mol% of FA species of G) TAG, H) FFA and I) DAG levels at 1% FBS with (grey) and without (red) TgGPAT. J) A ratio of the total PL content without/with TgGPAT in parasites under 0-1% FBS conditions and K) the same ratio of individual PL (phosphatidylinositol-PI, phosphatidylserine-PS, phosphatidylethanolamine-PE, lysophosphatidic acid-LPA, and phosphatidylcholine-PC) at 1% FBS, p-values are from an unpaired of the ratio vs 1 (unchanged levels). Percentage of13 C labelled FA species that are L) FASII-

Figure 5 :

 5 Figure 5: Lipidomic investigation of TgDGAT2A) A ratio of the total lipid content without/with TgDGAT2 in parasites under 0-10% FBS conditions, p-values are from an unpaired of the ratio vs 1 (unchanged levels). B) A ratio diacylglycerol-DAG (white) and triacylglycerol-TAG (black) without/with TgDGAT2 in parasites under 0-10% FBS conditions, p-values are from an unpaired of the ratio vs 1 (unchanged levels). Mol% of each FA species found in DAG at C) 0% and D) 1% FBS with (grey, -auxin) and without (red, +auxin) TgDAGT2. E) Immunofluorescence analyses of lipid droplet in parasites with and without auxin using nile red (in green) and Hoechst (DNA staining) under 0-1% FBS conditions, with an accompanying graph (left) with the average lipid droplet numb er per parasite quantified by iterative image processing. Mol% of each FA species found in FFA at F) 0% and G) 1% FBS with (grey, -auxin) and without (red, +auxin) TgDAGT2.

  Saharan Africa, South Asia and parts of South America. According to the World Malaria Report (2021), nearly half of the world's population is at risk of being infected with

	malaria (World Health Organization, 2021). Since 2015, the baseline of WHO's global
	malaria strategy, 24 nations have registered increases in malaria mortality. Due to
	service disruptions caused by the COVID-19 pandemic, emergence of drug resistance
	and vaccine inefficiency; critical 2020 milestones of WHO's global malaria strategy
	have been missed, and without immediate and dramatic action, the 2030 targets will
	not be met.

Malaria is caused by five species of Plasmodium parasites namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. The female Anopheles mosquito is

TABLE 1

 1

	TgACS Gene ID	Primers (gene specific sequence underlined)	Molecular system used

The patatin-like phospholipase PfPNPLA2 is involved in the degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development

	Nos données suggèrent que PfPNPLA2 contribue au maintien de la balances
	entre les phosphatidylglycérols et les acides lysobiphosphatidiques (mais pas
	les lysophosphatidylglycérol) au cours des phases du cycle asexué du parasite. Serena Shunmugam 1 , Yoshiki Yamaryo-Botte 1 , Ansgar Flammersfeld 2 , Sandra
	Nos données suggèrent également que cette enzyme est impliquée dans le Bennink 2 , Gabriele Pradel 2# , Cyrille Botte *1#
	recyclage des lipides du parasite, permettant la génération d'acides gras à
	partir de phosphatidylglycérol provenant des mitochondries ainsi que des 1. Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309,
	acides lysobiphosphatidiques. Ces acides gras recyclés peuvent ainsi être Université Grenoble Alpes, INSERM U1209, Grenoble, France
	utilisés pour la synthèse de phospholipides en conditions de stress nutritif. Ces 2. Division of Cellular and Applied Infection Biology, Institute of Zoology,
	observations RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany supportent l'hypothèse que la balance
	phospholipides/lysophospholipides et la disponibilité des nutriments sont # Equal contribution as senior authors
	essentielles au maintien du parasite en cycle asexué et/ou à leur passage au
	cycle sexué.
	• Malgré l'absence de changement des niveaux globaux de
	phospholipides, lorsque nous analysons les lipides individuels, nous
	observons :
	o Une augmentation des niveaux de phosphatidylglycérol
	o Une décroissance des niveaux d'acide lysobiphosphatidique
	sans modification des niveaux de lysophosphatidylglycérol.

• La séparation de lipides neutres par chromatographie sur couche mince montre que lorsque la protéine est déplétée : o Les niveaux de triglycérols sont systématiquement plus élevés o Les niveaux d'acides gras libres augmentent au stade annulaire mais décroissent au stade schizont. TITLE: * To whom correspondence should be sent to Cyrille Y. Botte; IAB UMR5309 CNRS INSERM UGA, Institut Jean Roget, Domaine de la Merci, 38700 La Tronche, France; (+33)4 76 63 71 23; cyrille.botte@univ-grenoble-alpes.fr / cyrille.botte@gmail.com

Table 1 :

 1 Primers used in this study

	Primer sequences (5'-3')
	PF3D7_1358000
					Amplified
	Cloning for pARL-	Fw			fragment
	HA-glmS	primer TAGCGCGGCCGCGGAAGAAGGCGATAAATTTGATCT	Restriction site: NotI	size:	bp
		Rev	TACGCCCTAGGTGTTTCTTTTTTAAAAAAGAAACATCTTAAT
		primer	AAGT	Restriction site: AvrII
	Integration-PCR of
	the KD
					Amplified
		Fw			fragment
	5' Integration	primer TAAGAAGAAGAAGAAATTAATTCCTCC		size:	bp
		Rev
		primer CCTTAGAGCTCGGCATAATCTGG
					Amplified
		Fw			fragment
	3' Integration	primer GCTTTACACTTTATGCTTCCGGCTCG		size:	bp
		Rev
		primer TTACTTTTTTTTAATATCATTTTTACGTCC

  /doi.org/10.1371/journal.ppat.1006362. Nolan, S.J., Romano, J.D. and Coppens, I. (2017b) 'Host lipid droplets: An important source of lipids salvaged by the intracellular parasite Toxoplasma gondii', PLoS Pathogens, 13(6), pp. 1-38. Available at: https://doi.org/10.1371/journal.ppat.1006362. Nolan, S.J., Romano, J.D. and Coppens, I. (2017c) Host lipid droplets: An important source of lipids salvaged by

	the	intracellular	parasite	Toxoplasma	gondii,	PLoS	Pathogens.	Available	at:
	https:/the	intracellular	parasite	Toxoplasma	gondii,	PLoS	Pathogens.	Available	at:

https://doi.org/10.1371/journal.ppat.1006362.

FUNDING SOURCES

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-FinoviAtip-Avenir Apicoplipid projects), CEFIPRA (Indo-French Centre for the Promotion of Advanced Research), IDEX-Université Grenoble Alpes, Region Auvergne Rhone-Alpes (IRICE Grant, Project GEMELI) and the Laboratoire d'Excellence ParaFrap, France (Grant number ANR-11-LABX-0024). The work was further funded by the priority programme SPP1580 of the Deutsche Forschungsgemeinschaft (GP). ACKNOWLEDGEMENTS/GRANT SUPPORT 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-FinoviAtip-Avenir Apicoplipid projects), CEFIPRA (Indo-French Centre for the Promotion of Advanced Research), IDEX-Université Grenoble Alpes, Region Auvergne Rhone-Alpes (IRICE Grant, Project GEMELI) and the Laboratoire d'Excellence ParaFrap, France (Grant number ANR-11-LABX-0024). The work was further funded by the priority programme SPP1580 of the Deutsche Forschungsgemeinschaft (GP).

Summary of published article

 [START_REF] Shunmugam | Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage[END_REF]:

The work described here sets the precedent of the basis of my thesis work and was initiated by Dr Sheena Dass. The lipid metabolism of apicomplexan parasites is highly dependent on combining fatty acids from different sources into lipid moieties described in the previous chapter. The flux of the multi-sourced FAs is governed by the nutrient availability found in the host cell and its environment. A fine balance between lipid synthesis and storage must be maintained for efficient parasite propagation. Our work

Sup. Figure 5: Supplementary lipidomic data of TgDGAT2-HA-mAID

A) A heatmap displaying a ratio of fatty acid (FA) levels in nmol/parasite ( 108 ) without/with TgDGAT2 in parasites under 0-10% FBS. B) Mol% of FA species of diacylglycerol (DAG) at 10% FBS with (grey) and without (red) TgDGAT2. Mol% of FA species of triacylglycerol (TAG) at C) 0%, D) 1% and E) 10% FBS with and without TgDGAT2. F) Lipid droplet analyses of the lipid droplet volume of this parasites line with and without auxin in 0-10% FBS. G) Mol% of FA species of free FA (FFA) this parasites line with and without auxin in 10% FBS. n = 3, unpaired t test p values are shown on graphs. Data are all presented as mean values +/-standard error of mean.

ABSTRACT RESUME

representative of standard error of mean (SEM) for each study. Statistical significance was determined for each experiment by t-test using GraphPad Prism (version 8).

Range of statistical significance was signified as per the p value, wherein 0.01-0.05=*, 0.01-0.001=** and <0.001=***.

SUPPLEMENTARY FIGURES:

Supplementary Figure 1: a) PCR confirmation of TgACS1-iKD clone 5 using the mAID system, showing correct integration of the mAID sequence, HX-drug cassette, and the gene-shift compared to the parental line. b) Western blot analyses showing down regulation of TgACS1 (100 kDa) after 30 mins of auxin treatment (100 µM), TOM40 (40 kDa) antibody used as loading controls. c) A replication assay of this line without (grey) and with (red) auxin revealed no effect on intracellular parasite replication. d) Relative mRNA expression of TgACS1 (compared to TgGAPDH) in intracellular (white) and extracellular (black) parasites in different FBS conditions. e)

The number of lipid droplets per intracellular parasite were not affected at 1% FBS but significantly reduced under complete nutrient loss (PBS), with (grey) and further decreased without (red) the protein of interest. f) A gliding motility assay showed that under only under complete nutrient loss (PBS, 2 h) and low nutrient conditions like 0% FBS, is the motility rate reduced without TgACS1 (red), in extracellular proteins. Supplementary Figure 2: a) Lipidomic profiles showing the mol% of each fatty acid comprising the total lipid fraction of intracellular 10% FBS without (grey) and with (red) auxin treatment in the TgACS1-iKD mutant line. b) Neutral lipid mol% of total lipid abundance in intracellular parasites in 10% FBS without and with auxin treatment, namely triacylglycerol (TAG), free fatty acid (FFA), diacylglycerol (DAG) and phospholipid (PL). Mol% of c) TAG and d) FFA levels in intracellular parasites at 0% FBS and e) TAG levels in intracellular parasites at 1% FBS. Mol% of f) TAG and g) FFA levels in extracellular parasites at 0% FBS. h) Lipidomic profiles showing the mol% of each fatty acid comprising the total lipid fraction of extracellular 10% FBS without (grey) and with (red) auxin treatment in the TgACS1-iKD mutant line. Neutral lipid mol% of total lipid abundance in extracellular parasites in 10% FBS without and with auxin treatment Supplementary Figure 3: TgACS1 does not rescue the growth of a yeast strain deficient in acyl-CoA activity of exogenous fatty acids. a) Wildtype yeast strain growth curves in a glucose (2%) and tween20 (0.2%, to induce β-oxidation) medium.

(Cer-, black, positive control to allow for FA synthesis), with the addition of 22.5 µM cerulenin only (Cer+, green, negative control), or additionally in the presence of 100 µM of FFAs C14:0 (yellow), C16:0 (blue) or C18:0 (red), respectively. b) Growth curves of a TgACS1 complemented faa1∆faa4∆ double yeast mutant in a glucose (2%) and Tween20 (0.2%) media supplemented with 22.5µM cerulenin and 100µM of FA as before. Medium with 22.5µM cerulenin but FFA was used as a negative control (black).

Summary of manuscript:

Characterization of Toxoplasma gondii acyl-CoA synthetases reveal the critical role of TgACS3 in adapting to the host nutrient environment

FA biosynthesis is crucial to lipid metabolism which is required for growth and division of apicomplexa parasites. This work is closely related to the work of TgACS1 found earlier in this chapter and will be submitted as 'back-to-back' articles. We looked to further characterize TgACS3, an essential protein for parasite growth that seemed to be most important at 10% FBS, or high host nutrient content.

The major findings of this study were:

•

TgACS3 is critical for tachyzoite intracellular development, especially when host nutrients are in excess To whom correspondence should be sent, cyrille.botte@univ-grenoble-alpes.fr / cyrille.botte@gmail.com; yoshiki.botteyamaryo@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 (FAs) 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. Previously 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. Now we show that the perinuclear/cytoplasmic TgACS3

Supplementary Figures

Supplementary Figure 1

Summary of submitted article:

The Here we look at a patatin-like phospholipase type A2 that is involved in phosphatidylglycerol metabolism.

The major findings of this study include:

gametocytes at 15' post-activation (p.a.) (Sup. Fig. 1B). Transcript analysis of the housekeeping gene aldolase was used as a loading control, and purity of the asexual blood stage and gametocyte samples was demonstrated by amplification of transcripts for the asexual blood stage-specific gene Pfama1

(apical membrane antigen 1) ( 36)and for the gametocyte-specific gene Pfccp2

(LCCL-domain protein 2) (37). The cDNA preparations lacking the reverse transcriptase were used to verify that samples were devoid of gDNA (Sup. Fig. 1B).

For functional characterization of PfPNPLA2, we generated a conditional PfPNPLA2-HA-KD line (using the pARL-HA-glmS vector) by fusing the sequences coding for a hemagglutinin A (HA)-tag to the 3´-coding region of Pfpnpla2 (Sup. Fig. 2A). In the PfPNPLA2-HA-KD, the Pfpnpla2 mRNA is expressed under the control of the glmS ribozyme, which catalyzes its own cleavage in the presence of GlcN and thus triggers transcript degradation.

Vector integration was confirmed by diagnostic PCR (Sup. Fig. 2B).

Immunoblotting of asexual blood stage lysate with an anti-HA antibody confirmed the synthesis of a HA-tagged PfPNPLA2 fusion protein in the untreated PfPNPLA2-HA-KD line, running at a molecular weight of 238 kDa (Fig. 1B). When the conditional knockdown of PfPNPLA2-HA synthesis was induced by treating asexual blood stage parasites with 2.5 mM GlcN for 72 h, PfPNPLA2-HA levels were reduced compared to the untreated control (Fig. 1B).

The untreated PfPNPLA2-HA-KD line was used for in-depth protein expression analysis. Furthermore, a HA-tagged PfPNPLA4 parasite line was generated, using the above mentioned pARL-HA-glmS vector. In addition, mouse antisera

PfPNPLA2 depletion differentially affects neutral lipid synthesis in ring and schizont stage parasites

To investigate the effect the loss of PNPLA2 has on overall parasite lipid metabolism, we conducted global lipidomic analyses using gaschromatography mass spectrometry on all asexual parasite stages (synchronized ring, trophozoite and schizont stages). It should be noted that despite PNPLA2 depicting essentiality in a lipid poor environment, all lipidomic experiments were conducted in normal culture conditions (with Albumax) as cultures did not survive long enough or yield a high enough parasitaemia when the protein was knockdown under limiting conditions.

We first sought to assess any potential defects at the total lipid content and then quantified major lipid classes of sustaining parasite survival, free FAs (building blocks of most lipids; FFA), diacylglycerols (major lipid precursors, DAG), PLs (major membrane lipids), and triacylglycerols (the main component of lipid storage bodies; TAG).

During ring stage development, the knockdown of PfPNPLA2 in the PfPNPLA2-HA-KD line had no significant effect on the global FA profile from the total parasite lipid content (Fig. 3A). However, further analyses, through separation of neutral and total PLs groups via 1D thin-layer chromatography (TLC), revealed significant increases in all lipid classes constituting neutral lipid content: TAG, FFA and DAG, whereas there was no impact on the total parasite PL content upon PfPNPLA2 knockdown (Fig. 3B). Further analyses to determine the FA composition of each lipid class showed that both FFA and DAG displayed significant increases in their C16:0 and C18:1 content (Fig. 3C-D), which are predominantly scavenged from the extracellular media (10,11).

Taken together, our results point to the importance of PfPNPLA2 to maintain proper parasite lipid homeostasis during blood stage replication, especially under low host nutrient content. It also points to the diversity and importance of the phospholipase activities that the parasite has developed and uses to maintain optimal asexual division and likely for other life stages. The number and diversity of them advocate for their importance and probable redundancy as well. Antibodies raised in this study against native putative phospholipases revealed differential localization of phospholipase, suggesting a redundancy/varied functionality (Fig. 1C-G). Phospholipase functional redundancy is well known and illustrated in Listeria monocytogenes, where mutation of a single phospholipase C resulted in moderate effects on mice infectivity and was severely enhanced when both were disrupted [START_REF] Smith | The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread[END_REF]. This interplay of phospholipases could be extrapolated to malaria parasites, but further gene-knock out studies are needed to understand the essentiality of this family of proteins.

We investigated 3 different putative patatin-like phospholipase enzymes, all which had different cellular localization and did not co-localize. This may suggest that each phospholipase has a non-redundant function. Further characterization of the other phospholipases must be done to prove this hypothesis.

DATA AVALIBAILITY STATEMENT

All data generated or analyzed during this study are included in this published article.

Summary of manuscript:

This work described my final thesis project which aims to characterize the cellular biochemical role of putative acyltransferases (AT), specifically glycerol-3-phosphate acyltransferase (TgGPAT) and diacylglycerol acyltransferase 2 (TgDGAT2). This project reiterates the metabolic flexibility of these parasites as the global theme of my thesis work. We developed two inducible lines of the above-mentioned proteins and there after conducted a series of experiments to determine their importance under different nutrient conditions.

The major findings of this study are:

• TgGPAT is the eukaryotic homolog of the apicoplast and plant-like TgATS1

• TgDGAT2 is previously uncharacterized and is not like TgDGAT1

• TgGPAT and TgDGAT2 are in close association with the endomembrane network of the parasites.

• TgGPAT is a critical protein needed for parasite development, which is responsible for lysophosphatidic acid (LPA) formation, which is pivotal to membrane biogenesis and neutral lipid synthesis.

• TgGPAT is essential under starved and excessive FBS conditions, unlike other ATs like TgAGPAT or TgATS2 (which are responsible for the second esterification of a FA to the glycerol-3-phoshate backbone forming phosphatidic acid)

• This suggests that GPATs' (TgGPAT and TgATS1) catalytic roles govern an indispensable reaction for parasite survival

• TgDGAT2 on the other hand, only seems important in low nutrient conditions and is crucial for lipid droplet (LD) biogenesis.

Toxoplasma gondii acyltransferases, TgGPAT and TgDGAT2, are involved in the balance between bulk membrane biogenesis and lipid storage dependent on host nutrient availability

Serena Shunmugam, Sarah Charital, Christophe-Sebastien Arnold, Samuel Duley, Nicholas J. Katris, Yoshiki Yamaryo-Botté*, Cyrille Y. Botté* Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, Université

Grenoble Alpes, INSERM U1209, Grenoble, France * Equal senior and corresponding authors. To whom correspondence should be sent, cyrille.botte@univ-grenoble-alpes.fr / cyrille.botte@gmail.com; yoshiki.botteyamaryo@univ-grenoble-alpes.fr

Abstract

Current evidence points at Apicomplexa parasites being highly flexible in adapting their metabolic program in response to nutrient availability from their hosts. Lipids are crucial nutrients for parasite division and hence parasite survival and pathogenesis.

These eukaryotic parasites can readily scavenge fatty acids (FAs) directly from their host but can also synthesize them de novo. Both pathways are crucial for the parasite,

Toxoplasma gondii strains and cultures

T. gondii tachyzoites (RH TIR1-3FLAG and inducible knock-down strains) were maintained in human foreskin fibroblasts (HFF) using Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies) supplemented with 1% foetal bovine serum (FBS) (Life Technologies), 2 mM glutamine and 25 µg/mL gentamicin at 37°C and 5% CO2.

Generation of inducible knockdown GPAT and DGAT2 lines in T. gondii

A C-terminally tagged HA line with inducible post-translational protein degradation was generated using the Auxin-inducible Degron system (AID). Homology regions of the C-terminal end of TgGPAT/TgDGAT2 excluding the stop codon were amplified using the primers found in Tables Table 1. Guide sequence for each protein (Table 1) were selected near the C-terminal end and was less than 100 bp away from at least one homology region. The guide sequence was inserted into Cas9 U6 universal plasmid Q5 mutagenesis or by enzyme digestion with BsaI and ligation.

The PCR product and the Cas-9 guide plasmid were used for transfection of a RH TIR1-3FLAG parental line [START_REF] Brown | Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii[END_REF]. Parasites were electroporated in a 2-mm cuvette using BTX ECM 630 (Harvard Appartus, at 1.100 V, 25Ω, and 25 µF).

After 24 h the newly transfected line was treated with 25 μg/ml mycophenolic acid and 50 μg/ml xanthine. Parasites were selected with the drug and cloned by limiting dilution. Screening of parasite clones for correct insertion of the mAID sequence, HX drug cassette and overall insertion/orientation were done with the Forward primer SSTg35 Reverse primers SSTg48, SSTg49 and SSTg38 for TgGPAT and SSTg39 for TgDGAT2 (Table 1). All PCRs were performed with TaKara primestar Max polymerase (Sup. Figure 2/3). The knockdown of these lines was induced with a final concentration of 100 µM of indole-3-acetic acid (auxin) made up in 100% ethanol.

Generation of a double mutant ∆DGAT1-DGAT2-iKD line in T. gondii

For the double mutant, we attempted to knock out (∆) TgDGAT1 in the TgDGAT2 mAID strain (above).

A guide sequence (Table 1) was selected near the within the gene sequence and was less than 100 bp away from at least one homology region (with the other homology region up stream of the start of the gene). The guide sequence was inserted into Cas9

U6 universal plasmid enzyme digestion with BsaI and ligation.

This ∆ strain was generated by amplification of a GFP signal sequence and a DHFRcassette (1 µg/ml pyrimethamine) from pDTG plasmid. This sequence was flanked by two homologous regions of TgDGAT1 on the 5' and 3' ends of this sequence. The primers used for this amplification are Table 1.

Transfection the TgDGAT2 mAID strain with the amplicon and U6 plasmid described for ∆TgDGAT1 was performed by electroporation as described above. After 24h, parasites were put under drug selection using 1 µM pyrimethamine.

Immunofluorescence

Primary antibodies anti-HA (rat, Roche), anti-CPN601 (rabbit), nile red were used at dilutions 1:250, 1:1000 and 1:1000 respectively. Secondary AlexaFluor 488-and 546conjugated anti-mouse/rat or anti-rabbit antibodies (Life Technologies) were used at 1:2000. For the immunofluorescence assay (IFA) parasites were grown on confluent HFF on coverslips and fixed in PBS containing 2.5% paraformaldehyde (PFA) for 15

Previous work by Quittnat et al., (2004), hypothesized TgDGAT1 to be essential due to failed attempts to knock-out this gene, granted in a time before CRISPR and other current advances in gene editing. We now show that TgDGAT1 can be deleted (Figure 3D) and that both paralogues seem to be non-essential for parasite survival in high lipid conditions (Figure C/D). Instead, we see that absent of both DGATs in this parasite under lipid scarcity leads to a favourable increase in parasite growth, this could suggest that these proteins are involved in controlling parasite growth when resources are limited. In this case the TAG levels are decrease (so LD cannot be formed) and we can hypothesize that accumulating FFA are instead channelled to bulk PL synthesis facilitating membrane biogenesis and division (7,31,32). We initially thought there would be function redundancy under these conditions (36), unlike in other eukaryotic models where DGAT1 and DGAT2 have preferences to exogenous and endogenous FAs, respectively (28,54). This can further be investigated by metabolic labelling studies. TgDGAT2 seemed to be crucial for parasite development at 0% FBS, imitating lipid starvation (Figure C), again suggesting another protein that is dependent on nutrient availability.

Using the TgDGAT2-mAID line, we were able to show that protein loss led to an increase in DAG levels, the substrate, but no changes in TAG levels were lipidomically observed. LD analyses showed that despite this the number of LD were diminished under TgDGAT2 loss (Fig. 5E), suggesting a crucial role in LD biogenesis. This process is highly dynamic and known to sequester several proteins for complex formation (55). Understanding this complex in apicomplexa versus humans, would be crucial in conceptualizing a novel therapeutic strategy against the parasites. This can be done by largescale protein association studies and biochemical techniques (MS-MS, NMR etc.).

The nutrient-dependency of these proteins provides insight into the parasite's nutrient flexibility in that they can adapt to different environmental conditions rendering proteins that were once non-essential, essential, or vice-versa.

Supplementary images

Sup. My PhD project focused on the processes involved in parasite nutrient adaptation when it comes to PL synthesis and lipid storage formation. I did this by investigating three protein families which I hypothesized are important in these mechanisms:

• Acyl-CoA synthetases (ACSs)

• Phospholipases

• Acyltransferases (ATs)

Deciphering the putative peroxisomal metabolome in Apicomplexa:

Our phylogenetic work now postulates that 7 ACSs exist in Toxoplasma parasites. A large scale full lipidomic/metabolic study of these highly diverse proteins would contribute to understanding the parasites complex lipid metabolism. I studied an unusual ACS, TgACS1, which can activate FAs derived from TAG under nutrient stress. This protein contains a peroxisomal targeting sequence (PTS), which has been characterized before as an essential tripeptide for correctly localization to the peroxisome (Ding, Clayton and Soldati, 2000;Kaasch and Joiner, 2000;[START_REF] Pratt | Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii[END_REF]