Claude Bernard -Lyon

M Frédéric Fleury

M Hamda

Ben Hadid

M Didier

M Pierre

Julia Mar´ıa

Coronas-Serna 1®

Arthur Louche

Mar´ıa Rodr´ıguez-Escudero

Morgane Roussin

Paul R C Imbert

Isabel Rodr´ıguez-Escudero

Laurent Terradot

Mar´ıa Molina

Jean-Pierre Gorvel

V´ıctor J Cid

Suzana P Salcedo
email: suzana.salcedo@ibcp.fr

Sarah Bigot

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism

Keywords: Pseudomonas, TIR domain, TLR adaptors, UBAP1, virulence Pull-down, Protein-protein interactions, Tagged protein, Affinity purification

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways

Remerciements :

Je tiens tout d'abord à remercier sincèrement ma directrice de thèse, le Dr Suzana Salcedo.

Suzana, ça fait maintenant un petit bout de temps que je travaille dans ton équipe de recherche. En effet j'ai commencé à travailler à ses côtés en Novembre 2013 où à l'époque elle m'avait engagé pour un contrat à durée déterminée de 1 an qui a été reconduit pour deux années supplémentaire en tant qu'assistant ingénieur/ ingénieur d'étude. A la fin de ces CDD, Suzana m'a proposé de réaliser une thèse dans son équipe de recherche, après avoir hésité j'ai finalement accepté de réaliser la thèse. Aujourd'hui je ne regrette absolument pas ce choix. Suzana a été une chef formidable, toujours à l'écoute et de bons conseils. Outre le coté scientifique et les journal club qui m'ont permis de développer un esprit critique, je la remercie également pour les moments partagés en équipe notamment les repas de Noël, les barbecues d'été et les activités que nous avons pu faire en dehors du labo. C'est un tout qui fait que les « plus ou moins » matin j'étais heureux de venir travailler au labo.

Je remercie l'Agence National de la Recherche pour leur financement. Je remercie Laurent Terradot, qui m'a accueilli dans son laboratoire pour mon stage de M2.

Laurent m'a appris toutes les étapes en partant de l'ADN jusqu'à l'obtention de protéine

Abstracts

Résumé en langue française :

Les agents pathogènes intracellulaires ont la capacité de moduler les réponses cellulaires de l'hôte pour survivre et proliférer. Ceci est réalisé en produisant et en injectant des protéines effectrices dans les cellules hôtes. Ces effecteurs peuvent notamment moduler la réponse immunitaire et le trafic intracellulaire. Brucella, l'agent causal de la brucellose, une maladie animale transmissible à l'homme, délivre des effecteurs dans la cellule hôte via son système de sécrétion de type IV. À ce jour, seuls quelques effecteurs ont été identifiés chez Brucella et leurs fonctions moléculaires restent floues.

Au cours de ma thèse, nous nous sommes intéressés à deux effecteurs de Brucella abortus contenant un domaine TIR, BtpA et BtpB, connues pour moduler la réponse immunitaire innée de l'hôte. Cependant, il a été démontré récemment que les domaines TIR purifiés ont une activité NADase in vitro. Nous avons ainsi montré que BtpA et BtpB conservent cette activité et sont capables de réduire la quantité de NAD présente dans la cellule hôte pendant l'infection. Ces résultats suggèrent que la NAD intracellulaire joue un rôle dans les infections à B. abortus et pourrait constituer un nouveau mécanisme de pathogénie. Parallèlement à ce projet, j'ai également eu l'occasion de travailler sur la caractérisation des effecteurs contenant un domaine TIR chez d'autres agents pathogènes. J'ai apporté une contribution majeure à la caractérisation de l'effecteur PumA chez Pseudomonas aeruginosa et participé à la caractérisation de l'effecteur TirS chez Staphylococcus aureus.

Le projet principal de ma thèse était d'initier une caractérisation structurale et fonctionnelle de deux nouveaux effecteurs de B. abortus, que nous avons nommés NyxA et NyxB. Après avoir établi qu'ils sont transloqués dans la cellule hôte pendant l'infection, nous avons observés ces effecteurs dans des compartiments nucléaires et cytosoliques. Par des expériences de co-précipitation, nous avons montré que ces deux effecteurs sont capables d'interagir entre eux et nous avons identifié SENP3 comme leur cible eucaryote. SENP3 est une déSUMOylase essentiellement située dans un sous-compartiment nucléaire, le nucléole. Cette protéine est impliquée dans la régulation de nombreuses fonctions cellulaires et joue un rôle majeur dans la biogenèse des ribosomes. Nous avons montré que SENP3 est nécessaire à la réplication de B. abortus dans la cellule hôte. L'analyse structurale de NyxB, nous a permis d'identifier une poche acide impliquée dans l'interaction avec SENP3. De façon surprenante, dans les cellules infectées par B. abortus, SENP3 est délocalisée et ne s'accumule plus dans les nucléoles. Dans les cellules transfectées par NyxA et NyxB, nous avons observé que ces deux effecteurs recrutent SENP3 dans un autre compartiment nucléaire : les corps nucléaires PML. En outre, ces effecteurs modulent également la distribution de NVL et RPL5, composants clés de la machinerie de biogénèse ribosomique dans les nucléoles, qui dans les cellules infectées forment des structures punctiformes dans le cytosol de manière dépendante de SENP3. Nous avons observé que ces structures cytosoliques correspondent à la localisation cytosolique des effecteurs Nyx et sont également enrichies en NUFIP1, un récepteur de la ribophagie.

En résumé, ce deuxième projet identifie deux nouveaux effecteurs chez B.abortus, qui ont un impact sur la localisation nucléolaire de SENP3. Ils sont impliqués dans la formation de structures cytosoliques enrichies en NUFIP1, NVL et RPL5, ce qui suggère un processus similaire à celui de la ribophagie.

Abstract in english :

Intracellular pathogens have the ability to modulate the hosts' cellular responses to survive and proliferate. This is achieved by producing and injecting effector proteins into host cells. These effectors can modulate various cellular functions, including immune response and intracellular trafficking. Brucella, the causative agent of brucellosis, an animal disease transmissible to humans, delivers effector proteins into the host cell through its type IV secretion system. To date, only a few effectors have been identified in Brucella and their molecular functions remain unclear.

During my thesis, we were particularly interested in two Brucella effector proteins containing a TIR domain, BtpA and BtpB, known to modulate the host innate immune response. However, more recently, purified TIR domains were also shown to have NADase activity in vitro. We have thus shown that BtpA and BtpB retain this activity and are able to reduce the amount of NAD present in the cell host during infection. These results suggest that intracellular NAD plays a role in Brucella infections and may constitute a new mechanism for Brucella pathogenicity. In parallel to this project, I also had the opportunity to work on the characterization of TIR-domain-containing effectors in other pathogens. I have made a major contribution to the characterization of the PumA effector in Pseudomonas aeruginosa and participated to the characterization of the TirS effector in Staphylococcus aureus.

The major project of my thesis was to initiate a structural and functional characterization of two new B. abortus effectors, which we named NyxA and NyxB. After establishing they were translocated into the host cell during infection, we imaged these effectors and found them in nuclear and cytosolic compartments. By pull-down experiments, we showed that these two effectors were able to interact with each other and we identified SENP3 as their eukaryotic target. SENP3 is a deSUMOylase essentially located in a nuclear sub-compartment, the nucleolus. This protein is involved in the regulation of many cellular functions and plays a major role in ribosome biogenesis. We have shown that SENP3 is necessary for the replication of B. abortus within the host cell. The structural analysis of NyxB allowed us to identify an acidic pocket involved in the interaction with SENP3. Surprisingly, in cells infected with B. abortus, SENP3 is delocalized and no longer accumulating in nucleoli. In NyxA and NyxB transfected cells we could observe these two effectors recruited SENP3 to another nuclear compartment: the PML nuclear bodies. Furthermore, these effectors were also able to modulate the distribution of NVL and RPL5, key components of the ribosomal biogenesis machinery in the nucleoli, which in infected cells form cytosol punctate structures in a manner dependent on SENP3. We have observed that these cytosolic structures correspond to the cytosolic location of Nyx effectors and are also enriched in NUFIP1, a ribophagy receptor.

In summary, this second project identifies two new Brucella effectors, which have an impact on the nucleolar localization of SENP3. They are involved in the formation of cytosolic structures enriched in NUFIP1, NVL and RPL5 suggesting a process similar to that of ribophagy. Brucellosis is a worldwide infectious disease caused by intracellular bacteria belonging to the genus Brucella [1]. It seems that brucellosis is an old disease, present among humans since a long time. Indeed, macroscopic and microscopic analysis of the lumbar vertebrae of ancient human remains of the Pliocene period (2.8 million years ago) have been suggested as signs of brucellosis [2]. Archaeologists have also found a jar containing fossilized cheese in an Egyptian tomb dating back to the 19th dynasty (-1293(- -1185 BC) BC), for which proteomic analysis revealed the presence of Brucella [3]. Moreover, symptoms reminiscent of brucellosis have been described by Hippocrates in his publication Epidemics (around 410 BC). It wasn't until the late 19 th century that the causative agent of brucellosis was isolated by a British physician Dr David Bruce from the spleen of a dead soldier on the Malta island, and for this reason the organism bears his name. Over time brucellosis had different names, including undulant fever, Maltese fever, Gibraltar fever, Crimean fever, goat fever and Bang disease.

Epidemiology

Brucellosis in the world:

Brucellosis is one of the most prevalent zoonosis. The word zoonosis, from the Greek zoo (animal) and nosos (disease) are defined as diseases that are naturally transmitted from vertebrate animals to humans. Every year more than 500,000 cases of human brucellosis are recorded [4], which is why WHO has classified this disease as an important neglected zoonosis re-emerging in the world. However, the incidence of disease is believed to be under-diagnosed and the true incidence is evaluated to be 10 times higher (5,000,000 cases) [5]. Brucellosis is still endemic in many regions, namely the countries of the Mediterranean basin, Latin America and the Middle East. Throughout the world, the geographical distribution of the disease varies greatly (figure1). While the incidence of the disease is clearly declining in the developed world, this is not the case in developing countries where it often reaches worrying levels. The prevalence of the disease ranges from 0.3 cases per million in developed countries to more than 1000 cases per million in endemic regions [6].

Brucellosis in France:

As a result of strong prophylactic campaigns in the 1960s on ruminant farms, cases of human brucellosis in France decreased from 800 cases in 1976 to 44 cases in 2000 [6]. France has been declared officially free of bovine brucellosis since 2005, and no outbreaks in animals have been identified on the national territory from 2003 to 2012. Every year, cattle, sheep and goat farms are regularly tested for brucellosis. Now, any confirmation of brucellosis on a farm will lead to slaughtering of the entire herd and the destruction of resulting products, as a precautionary measure.

However, two outbreaks of bovine brucellosis were confirmed in 2012 on the French territory, thus calling for vigilance regarding this zoonosis [7] [8]. A first outbreak was due to the importation of an infected bovine animal and was rapidly eradicated. The second outbreak was related to a large wild reservoir discovered in mountain ibexes [8], which resulted in a The most affected countries are Mongolia and the countries of the Middle East and the Balkans. Some Central and South American countries and the countries of the Mediterranean basin also have numerous cases of human brucellosis. [6] massive eradication program of the older ibex members of the herd (infected and noninfected) in the Bargy mountain range to prevent further dessimination. These measures were highly controversial and in fact proven inefficient in the control of brucellosis in the ibex population. Therefore, recent new surveillance measures have been set in place for monitoring all wild-life in the region (ibex, chamoises, deer) as well as local pastures (sheep, goats and cows) [9]. Selective slaughtering programs are in place and a vaccination program being studied.

Brucella spp

Brucella are small coccobacilli Gram-negative bacteria, 0.6-1.5 μm long by 0.5-0.7 μm in width. They are non-motile, non-encapsulated, non-spore-forming and do not present pili nor toxins. Brucella are strictly aerobic but some strains growth better in an atmosphere containing 5 to 10 % CO2. Brucella does not present classical virulence factors known in others pathogenic bacteria such as: lysogenic phages, virulence plasmids, invasive protease, exotoxin and toxic lipopolysaccharide (LPS).

Nevertheless, the LPS plays an important role in the virulence of Brucella. The LPS, which found in the outer membrane of all Gram-negative bacteria, is composed of a hydrophobic lipid moiety, called lipid A, normally associated with its toxic properties linked to a polysaccharide, with a hydrophilic core and a repeating O-antigenic oligosaccharide side chain.

There are 2 types of LPS depending on the surface morphology: smooth or rough. Smooth correspond to the presence of the O-polysaccharide side chain in the LPS moiety and rough without the O-polysaccharide side chain. Most Brucella species are naturally smooth, except for B. ovis and B. canis but can transition to form rough colonies in the laboratory, usually associated with reduced virulence.

Bases on 16S rRNA sequence homology, Brucella spp. belongs to the α2-subdivision of the proteobacteria, together with animal pathogens such as Bartonella spp., Rickettsia spp. and plant pathogens such as Agrobacterium tumefaciens, but also plant symbiont such as Sinorhizobium meliloti [10].

Brucella spp. are facultative intracellular bacteria that can infect many species of animals and can also be associated with disease in humans. Recently, identification of several new Brucella species has considerably expanded the genus. To date 12 species form the genus Brucella. The classification of these species was based on the difference in host preference (Table 1) and phenotypic characteristics. In 1968, the genus was composed by 6 "classical" or "core" Brucella species affecting terrestrial mammals: Brucella abortus, Brucella canis, Brucella melitensis, Brucella neotomae, Brucella ovis and Brucella suis [4]. These species are very close genetically, they share on average more than 94% of identity at the nucleotide level [11].

Biovars were recognized for certain of these species. Since the 1990s, 6 news and "atypical" strains have joined the genus Brucella: Brucella microti, Brucella inopinata, Brucella ceti, Brucella pinnipedialis, Brucella papionis and Brucella vulpis.

Each species of Brucella has one or more preferential animal reservoirs that maintain their transmission cycle (Table 1). However, they are not totally specific to their reservoir.

Recently, bacteria of the genus but not belonging to any of the known families have been isolated in amphibians as the natural host [12], the first non-mammal host described for Brucella. In vitro and in vivo experiments suggest that this strain is able to invade and proliferate in macrophages but also survive in the murine host model. The mechanisms explaining the apparent Brucella host-specificity of the different species remains to this day poorly understood. The majority of brucellosis infections in humans are due to: Brucella melitensis, Brucella abortus, Brucella canis and Brucella suis (Table 1). In addition, the new strains of Brucella ceti and Brucella pinnipedialis associated with cases of brucellosis in marine mammals also appear to be capable of transmitting the disease to humans [13].

Table 1: Host preference and zoonotic potential of the different Brucella species. Modified from [14]. Rough [16]

Brucella

Brucella neotomae

Desert wood rat

No reported infection Smooth [17]

Brucella pinnipedialis

Pinnipeds (Seals, sea lions and walruses)

Low

Smooth [18] Brucella ceti Cetaceans (Dolphins, whales, porpoises)

Low Smooth

Brucella microti

Wild voles, red foxes No reported infection Smooth [19]

Brucella inopinata

Unknown Human from a breast implant infection, Amphibians

High Smooth [20] Brucella papionis

Baboons

No reported infection (?) [21] Brucella vulpis Red foxes No reported infection (?) [22]

Transmission

In animals, transmission occurs generally at the time of abortion or parturition. High concentrations of Brucella are found in fetal waters from infected animals [23]. This pathogen is able to survive outside its host for up to several months depending on the environment, temperature, humidity.

Human brucellosis has always been associated with an animal reservoir of Brucella spp. [24]. We can distinguish different transmission routes:

-The most common way is the ingestion of food from animal origin, unpasteurized milk or raw dairy products. Indeed, when animals are infected with Brucella, their milk becomes contaminated. Consumers of unpasteurized dairy products are at risk of contracting brucellosis. Nevertheless, veterinary controls and pasteurization have made contamination by dairy products exceptional in France.

-Inhalation of aerosolized infectious material is one of the most common routes for human infection. Persons at risk for this mode of transmission are meat slaughterhouse workers and people in laboratories that work with the bacteria [25]. Brucellosis is the most common laboratory-acquired infection, in the majority of cases aerosols are involved [26] [27].

For example, more than 100 students and staff became infected in two different institutes in China (Lanzhou and Harbin Veterinary research institute) (Nature new 17december2019).

That is why in French laboratories, experiments on Brucella must be carried out in a biosafety level 3 (BSL3) containment. Its high infectious power (10 to 100 bacteria are sufficient to infect humans) coupled with its possible spread by aerosols make Brucella an organism with the potential to be used as a weapon of bioterrorism [28]. For this reason, Brucella has been included in the list of possible bioterrorism agents by the Centers for Disease Control and Prevention (CDC) [6].

-Close contact with infected animals. Brucella can penetrate into the body through skin wounds or mucous membranes. People most sensitive to this type of transmission are veterinarians, hunters and slaughterhouse workers.

-Human to human transmission is very rare. The disease can be transmitted to the newborn through transplacental route or breastfeeding of the infected mother.

Transplantation, sexual or blood transfusions have also been reported as possible routes of human to human transmission [29] [30] [31].

Symptoms

Symptoms are different between animals and humans.

In animals, Brucella has a tropism for the reproductive system. In pregnant female animals, bacteria colonize the placenta leading generally to abortion of the foetus in the second half of gestation. This is due to high colonization of trophoblasts by Brucella. Brucellosis in cows and goats also leads to a reduction in milk production, due to bacterial colonization of the mammary glands [32] [33].

In male animals, brucellosis is characterized by orchitis which leads to sterility. In both male and female animals, symptoms can include arthritis [34].

In humans, brucellosis is a serious debilitating disease that can have a fatal outcome if untreated. The incubation period of the pathogen is 1 to 4 weeks but sometimes the incubation period can last several months without any symptoms. The symptoms of brucellosis are not dependent on the mode of transmission by which the patient became infected.

The clinical presentation of brucellosis can be subdivided in three phases: acute, sub-acute and chronic. In the acute phase, clinical manifestations are variable and non-specific. The infected individual may present with flu-like symptoms, fever, chills, sweating, malaise, weight loss or osteoarticular pain, hepatosplenomegaly, lymphadenopathy and hearing loss [35].

Then the bacterium spreads and can reach different organs. In absence of treatment, the disease evolves into a subacute or chronic phase which are much more complicated to treat [36]. The sites of infection are the bones and joints, the liver, and sometimes the heart and the nervous system. The latter two infections cause endocarditis and neuro-brucellosis, which can lead to death in 2% of cases [37].

Diagnosis

Diagnosis from clinical observation data is difficult due to symptom variability and nonspecificity in the acute phase. Nevertheless, it is important to be able to start as early as possible a treatment in order to avoid the establishment of the chronic phase. For this reason, diagnosis is initially based on the patient's history of possible exposures (travels and/or consumption of contaminated milk products imported from endemic areas…). Subsequently, an adequate laboratory diagnostic method is essential to confirm or not brucellosis. There are several diagnoses that can be direct or indirect.

Direct diagnosis includes culture and isolation of the strain from biological samples (blood culture, lymph node, bone marrow, cerebrospinal fluid…), which is the gold standard.

However, in the chronic phase of the disease, detection is only very rarely positive in the case of blood cultures. However, this technique is time-consuming and expensive. Another direct detection technique is polymerase chain reaction (PCR) and real-time PCR. These techniques are sensitive and specific. They also have the advantage of being relatively fast and reduce the risk of acquisition of brucellosis by the personnel performing the analysis, because the bacteria are inactivated. On the other hand, false negatives may be observed as a result of the presence of DNA polymerase inhibitors in clinical samples. False positives may also be observed in the case of sample contamination or cross-amplification reactions.

Indirect diagnoses include serological tests, which are the most commonly used diagnoses for brucellosis. They have the advantage of being quick, less expensive and safer. Among these techniques are the Rose Bengal Test, the Wright agglutination (WHO reference reaction), the Coombs antibrucella, immunocapture techniques and serology to detect specific IgG and IgM antibodies. False positives may occur due to an antigenic relationship with other pathogens (Escherichia coli O157, Francisella tularensis, Yersinia enterocolitica, Vibrio cholerae and Salmonella species) [38].

Treatments, prevention & vaccins

Unfortunately, there is no safe and effective vaccine for brucellosis in humans. During the acute phase, the treatment of brucellosis is based on the combination of two antibiotics doxycycline and rifampicin (treatment according to WHO) for a period of 6 weeks or streptomycin and doxycycline [39]. However, in some cases relapses are reported, despite the treatment [40]. In the absence of treatment or when treatment is insufficient, the disease progresses and enters a subacute focused phase, leading to the constitution of isolated or multiple infectious foci at the osteoarticular, neurological, genital, hepatic or cardiac level. In this case a treatment based on three antibiotics (doxycycline, rifampicin and gentamicin) is used at least 3 months. In the chronic phase, treatment of the disease is made impossible by the fact that the bacteria have become inaccessible. Only symptoms are treated, sometimes surgical operations are performed to remove the site of infection in the case of endocarditis or osteoarticular localization.

Prevention of infection relies on reducing people's exposure to infected animals, food hygiene measures (milk sterilization), personal hygiene measures (wearing gloves, masks) for exposed occupations. In order to be able to control the disease in the human population it is important to be able to control and eradicate the disease in domestic and wild animals. Indeed, the decrease in the incidence of the disease in animals is correlated with a decrease in the number of infected individuals. Control and eradication of the disease in animals must be achieved through surveillance tests, slaughter of infected animals but also vaccination. Indeed, vaccination of cattle, sheep and goat herds is one of the best means of prevention to limit the transmission of the disease as much as possible. There are two types of brucellosis vaccines:

live attenuated vaccines and inactivated vaccines. Currently the vaccines licensed for use in livestock are live attenuated vaccines. The most commonly used vaccines to protect farm animals are S19 and RB51 for Brucella abortus, Rev1 for Brucella melitensis.

Vaccine strain S19 is a live B. abortus, spontaneously attenuated mutant discovered in 1923 (Graves., 1943). This vaccine has been the reference vaccine and has a high level of protection.

It is used for the vaccination of calves and cows with reduced dose. This vaccine provides a good rate of immunization and greatly reduces the number of abortions. However, S19

vaccination has some disadvantages. S19 has a smooth phenotype due to its O-antigen LPS which induces a specific antibody response and leads to interference with serodiagnosis [41].

This serological interference impedes the distinction between immunized and naturally infected animals. It has a residual virulence that can lead to abortion if administrated in pregnant cows, and orchitis in male. For this reason, vaccination with S19 is currently only available in female calves between 3 and 8 months of age. In addition, S19 is fully pathogenic to humans, so veterinary personnel should pay particular attention at the time of vaccination to avoid accidental contamination.

Vaccine strain RB51 is a live attenuated B. abortus developed in 1982 [42]. Unlike S19, RB51 has a rough phenotype, it does not possess O-antigen LPS chain and therefore does not present serological interference problems. Like S19, RB51 has a good protection against infection and abortion [43]. For this reason, it is used as the reference vaccine in many countries. However, this vaccine does have some drawbacks. RB51 is still pathogenic for humans and it is resistant to rifampicin which is use in treatment in human brucellosis.

Vaccine strain Rev1 is a live attenuated B. melitensis developed in the 1960s. To date Rev1 is the most effective strain against goat and sheep brucellosis [44]. Just like S19, Rev1 has a smooth phenotype and therefore will cause serological interference problems and is still virulent to humans [45]. In pregnant animals, Rev1 can lead to abortion even with reduced doses of vaccine.

Efforts are currently being made to develop new and better livestock vaccines, and generate human vaccines. Live attenuated vaccines against farm animals have been in use for several decades. They are mostly effective in preventing abortion and disease transmission. However, they still have disadvantages as described above and immunization protocols must be chosen carefully (single or multiple dose vaccination, reduced dose, route of administration, age of the animal to be vaccinated, etc). Besides, not all countries have the same vaccination regulations. In France, for example, vaccination of cattle, sheep and goats is prohibited because vaccination is not compatible with serological screening. However, departments that are still infected may have derogations for the vaccination of sheep and goats.

Consequences

Nowadays, this disease seems to be re-emerging in the world, with the identification of new strains (marine mammals, and amphibian) and the appearance of wild foci (bison, deer, hare, caribou, wild boar, chamois, ibex, etc.). Indeed, animal brucellosis in wild animals constitutes a Brucella reservoir with possibility of accidental transmission to farm animals and therefore also to humans [46] [47]. This is case in the Greater Yellowstone in USA where bison and elk remain a major reservoir for the disease [48] or in France with ibex and wild boar [49].

Brucellosis is still a highly infectious disease with a worldwide distribution. It is a threat to public health and also has important socio-economic repercussions (along with farmer emotional suffering). The deficit in animal production due to reduced milk production, abortion, and slaughtering of infected animals [50] leads to important economic losses in developing countries estimated to hundreds of millions of dollars [51]. Brucellosis control measures have proven to be effective since many developed countries are now considered free of brucellosis. Nevertheless, efforts must be focused on educating the population in order to acquire the barrier gestures and on the scientific community in order to better understand the mechanisms of virulence of the bacterium with the aim of developing more effective vaccines.

Brucella intracellular life

Like Legionella, Listeria and Salmonella, Brucella are classified as facultative intracellular pathogens, in contrast to obligate intracellular pathogens such as Chlamidia, Rickettsia or

Coxiella. However, the term facultative extracellular pathogen might be more appropriate, as to date no environmental reservoirs have been identified and the intracellular environment is suitable to Brucella replication [52]. Indeed, Brucella is extremely well adapted to intracellular life style, it is able to manipulate intracellular traffic, immune response, and replicate within host cells without inducing cell death. Furthermore, hiding within eukaryotic host cells limits exposure to immune responses, competition with other microbes and protects Brucella against the effect of antibiotics making treatment more complicated.

This pathogen is able to enter, survive and replicate in a wide range of mammalian cell types, including professional phagocytes such as macrophages and dendritic cells and nonprofessional phagocytes such as epithelial cells, fibroblasts and trophoblasts [53] [54] .

Entry into the cells

Successful bacterial invasion depends on two consecutive steps: binding and internalization.

The mechanism of attachment and entry into cells host by Brucella are not fully understood and is still controversial.

In non-professional phagocytic cells, Brucella is taken up within minutes after inoculation, with one or two bacteria per cell [55]. In this process, lipid-rafts are involved.

Brucella's entry into the host cell is abolished by inhibiting clathrin, suggesting it plays a fundamental role in the entry [56]. Brucella uptake by HeLa cell leads to a slight reorganization of the membrane with enrichment of the F-actin cytoskeleton at the entry site. Previously it has been shown that infection performed in the presence of cytochalasin D and colchicine, two specific inhibitors of microfilaments and microtubules respectively also hamper internalization [START_REF] Guzmán-Verri | GTPases of the Rho Subfamily Are Required for Brucella abortusInternalization in Nonprofessional Phagocytes[END_REF].

Rho small GTPases (Rho, Rac and Cdc42) known to regulate the organization of actin cytoskeleton, are required for actin polymerization and Brucella penetration into cells. Cdc42

is directly recruited and activated by virulent Brucella [START_REF] Guzmán-Verri | GTPases of the Rho Subfamily Are Required for Brucella abortusInternalization in Nonprofessional Phagocytes[END_REF]. Regulator BvrR and sensor protein BvrS coding by the two-component regulatory system BvrR/BvrS are involved in the invasion of Brucella in both professional and non-professional phagocytic cells. Brucella BvrR/BvrS mutants fail to recruit small GTPases of the Rho subfamily [58] [59].

In professional phagocytic cells, two cases can be distinguished depending on the opsonic condition of the bacteria. In the case of opsonized Brucella, internalization occurs via complement and Fc receptors. After recognition by the receptor, Brucella is internalized by phagocytosis which occurs by a zipper-like mechanism. In this case, Brucella is mostly phagocytized by activated macrophages which can efficiently destroy bacteria [52].

In the case of non-opsonized Brucella, internalization into host macrophages is mediated by components of lipid rafts such as cholesterol and ganglioside GM1 on the cell host plasma membrane [START_REF] Watarai | Macrophage Plasma Membrane Cholesterol Contributes to Brucella abortus Infection of Mice[END_REF] [61] [START_REF] Porte | Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages[END_REF]. In this case, internalization is dependent on toll like receptor 4 (TLR4) and PI3 kinases. Indeed, when TLR4 is disrupted internalization of Brucella abortus is suppressed [63] [64,65].

The LPS also plays an important role in bacterial adhesion and internalization. In smooth strains, the LPS O side chain interacts directly with the class A scavengers receptors (SR-A) present in lipid rafts of host cell plasma membrane [START_REF] Kim | Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus[END_REF]. Macrophages from SR-A-deficient mice hamper Brucella internalization and replication, showing internalization is SR-A dependent. Nevertheless, signal transduction mediated by SR-A remains unknown. In contrast, rough strains, whose LPS does not have O side chain, do not enter through lipid rafts and are quickly degraded by lysosomes [START_REF] Porte | Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages[END_REF].

Another receptor that has been shown to be involved in the internalization of Brucella abortus in macrophages is the cellular prion protein (PrPc) present on the surface of macrophages or M cells through Hsp60 a chaperone of the GroEL family [67] [68]. However, these studies remain controversial [START_REF] Fontes | Absence of evidence for the participation of the macrophage cellular prion protein in infection with Brucella suis[END_REF].

To enable adhesion to cells Brucella has developed surface molecules that target different cellular receptors. In HeLa cells as well as macrophages, Brucella adhesion is mediated by SP41 a surface marker of the brucellae which interact with eukaryotic receptors containing sialic acid [70] [71]. More recently, additional adhesins have been described, including BigA [START_REF] Czibener | BigA is a novel adhesin of Brucellathat mediates adhesion to epithelial cells[END_REF] and the trimeric autotransporter BtaE [START_REF] Ruiz-Ranwez | an Adhesin That Belongs to the Trimeric Autotransporter Family, Is Required for Full Virulence and Defines a Specific Adhesive Pole of Brucella suis. Payne SM[END_REF].

Intracellular traffic

Once inside phagocytic or non-phagocytic cells, Brucella is found in a membrane-bound compartment, the so-called Brucella-containing vacuole (BCV). These BCVs traffic along the endocytic and secretory pathway and mature over time (figure 2).

The initial stage of trafficking take place between 0-and 8-hours post-infection and the BCV is called endosomal BCV (eBCV). During this stage, eBCV fuses with early endosomes and acquires some of their markers such as the small GTPase Rab5 and Early endosome Antigen 1 (EEA1). Thereupon, eBCV partially fuses with late endosomes and lysosomes where it acquires late endosomal markers such as Lysosomal-Associated Membrane Protein 1 (LAMP-1) as well as the small GTPaseRab7 [START_REF] Starr | Brucella Intracellular Replication Requires Trafficking Through the Late Endosomal/Lysosomal Compartment[END_REF]. This transient fusion with the lysosome lead to an acidification of the BCV reaching a pH of 4.5 [START_REF] Porte | Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages[END_REF]. eBCV acidification is essential for correct traffic and replication in macrophages and HeLa cells during early stage of infection [START_REF] Porte | Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages[END_REF].This acidification triggers the expression of the VirB type IV secretion system (T4SS) [START_REF] Lacerda | Brucella T4SS: the VIP pass inside host cells[END_REF]. T4SS allows the translocation of several bacterial effector proteins into the cell host to modulate various cellular processes including BCV trafficking and host immune responses, which we will be discuss in the corresponding T4SS section. Several studies have shown that replication of Brucella in both macrophages and epithelial cells as well as in vivo models of infection requires the expression of the T4SS [52]. Additional virulence factors have also been implicated, such as the cyclic -1,2-glucan (CG) synthesized by Brucella that is important for preventing total fusion with lysosomes and thus degradation [START_REF] Arellano-Reynoso | Cyclic β-1,2-glucan is a brucella virulence factor required for intracellular survival[END_REF]. It is important to note that at this early trafficking step, more than 90% of bacteria are killed inside the BCV in macrophages, meaning that only a few of the internalized bacteria are able to reach their replicating niche.

The endoplasmic reticulum: the replicating niche

Between 8 and 12 hours post-infection, eBCVs are redirected towards the endoplasmic reticulum (ER) and interacts with the Endoplasmic Reticulum Exit Sites (ERES). eBCVs lose the endosomal markers and acquires some specific markers of the ER such as calnexin, calreticulin, Sec61β [78] [54], giving rise to replicative BCVs (rBCV). This interaction is regulated by the small GTPase Sar1 involved in the formation of the COPII complex. This complex allows shuttling between the membranes of the ER towards the Golgi apparatus.

Inhibition of Sar1 activity blocks replication, indicating that this interaction is essential for the establishment of the Brucella replication niche [START_REF] Celli | Brucella coopts the small GTPase Sar1 for intracellular replication[END_REF]. There is also other evidence that rBCVs interact with the vesicular traffic between the ER and the Golgi apparatus. Rab2 and the GAPDH are recruited to the BCV. These two proteins are involved in the vesicular transport between the ER and the Golgi apparatus [START_REF] Fugier | The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication[END_REF]. Brucella effectors secreted by the VirB T4SS and that are involved in this process have been identified [81] [78] [82]. Studies have identified several effector proteins targeting host secretory functions among them, BspA, BspB and BspF that contribute to bacterial replication by impairing host secretory trafficking [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF] and RicA [START_REF] De Barsy | Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2[END_REF]. For example, BspB interacts with the Golgi apparatus-associated conserved oligomeric Golgi (COG) complex which is a key regulator of vesicular traffic at the Golgi apparatus [START_REF] Miller | A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication[END_REF].

This conversion into rBCV, provides a suited environment for Brucella replication.

Interestingly, initiation of bacterial division occurs at the eBCV stage, suggesting sensing of changing conditions within the vacuole [START_REF] Deghelt | On the link between cell cycle and infection of the Alphaproteobacterium Brucella abortus[END_REF]. Indeed, it has been clearly shown that Brucella cell division is intimately connected to intracellular trafficking [START_REF] De Bolle | Brucella abortus Cell Cycle and Infection Are Coordinated[END_REF]. During the first stage of BCV maturation, the bacterial cell cycle is arrested. However, before conversion of eBCV to rBCVs, bacterial division is initiated, which then likely assists the action of the T4SS and enables further maturation of BCVs into a compartment suited for proliferation.

Once the intracellular niche of replication is established, the bacteria aim to remain in the cell for as long as possible by inhibiting apoptosis [88] [89]. Thereupon between 12 and 48 hours post-infection, Brucella extensively proliferate in rBCVs to occupy almost the entire cytoplasmic volume of the host cell.

The exit of Brucella : The autophagic BCV

It has been proposed that Brucella completes its intracellular cycle by the formation of autophagosome-like structures, autophagic BCVs (aBCVs) [START_REF] Starr | Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle[END_REF].

Autophagy is a ubiquitous degradation mechanism, orchestrated by more than 30 specific proteins called ATG (for autophagy-related gene) in a multi-step process, involving at least three main phases: Initiation, nucleation and elongation. It is a dynamic membrane process that begins with the de novo formation of vacuoles called autophagosomes encompassing cytoplasmic fractions into a double-membrane compartment. This process allows the capture of aggregated/misfolded protein, damaged organelles or pathogens. Subsequently these autophagosomes fuse with the lysosomes where their contents will be degraded providing nutrients or elimination of the pathogen. Although it has a role against microbial invasions, all or part of the autophagic process can be blocked, or hijacked, by microorganisms for their own benefit [START_REF] Huang | Bacteria-autophagy interplay: a battle for survival[END_REF].

As a result of rBCV proliferation, strating at 48 hours after infection, BCVs lose ER markers and gain features consistent with an autophagosome. The formation of these vacuoles, named aBCVs for autophagic BCVs, is dependent on nucleation proteins such as Beclin1, ULK1 and Atg14L, but independent on elongation proteins Atg5, Atg7, Atg4 or Atg16L [START_REF] Starr | Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle[END_REF]. Thus, Brucella is able to subvert part of the autophagy machinery by modulating specifically autophagy initiation/nucleation complexes to allow its exit from the host cell and the infection of new adjacent cells.

Figure 2. Brucella intracellular lifecycle

Once internalized by host cells, Brucella is contained in a vacuole named Brucella-containing vacuole (BCV). Brucella will follow the endosomal pathway and mature over time. BCV interacts with the host cell to first become endosomal BCV (eBCV), then replicative BCV (rBCV) and finally autophagic BCV (aBCV). [START_REF] Celli | The Intracellular Life Cycle of Brucella spp[END_REF].

Brucella T4SS and its effectors

Pathogenic bacteria are often able to secrete macromolecules necessary for virulence in the extracellular medium but also export them into host cells. These macromolecules must be able to cross the internal and external membranes of the bacteria, which are naturally hydrophobic and are therefore impermeable to hydrophilic macromolecules. Dedicated secretion systems make it possible to transport these macromolecules through the bacterial membranes and have an essential role in bacterial pathogenesis. Nine secretion systems are currently known in Gram-negative bacteria: types I to IX.

Brucella T4SS

In 1999, thanks to the sequencing of the Brucella suis genome, O'callaghan et al. identified an operon coding for a type IV secretion system homologous to the virB system of Agrobacterium tumefaciens [START_REF] O'callaghan | A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis[END_REF]. The virB operon is conserved in all the sequenced Brucella species.

 T4SS functions  Type 4 secretion systems are protein complexes that carry out the transfer of macromolecules (protein, DNA) across the two bacterial membranes. They are found in several pathogenic bacterial species such as Agrobacterium, Legionella, Bartonella, Bordetella, Helicobacter and Brucella. T4SS have several functions, namely: i) the transfer of an effector molecule from the pathogen to the host cell, ii) the release of DNA allowing the exchange of DNA with the extracellular medium, iii) bacterial conjugation, which ensures the transfer of DNA to a target receptor cell allowing the acquisition of a selective advantage [94] [95].

 T4SS architecture  The best characterized T4SS to date is the VirB/D4 system from A. tumefaciens. VirB/D4 is composed of 12 proteins called VirB1-11 and VirD4. This system serves as a reference for the study of effector translocation by T4SS in Gram-negative bacteria. The VirB/D4 system forms a dynamic protein complex consisting of three functional groups of proteins: i) the pilus, present on the bacterial surface, is formed by the VirB2 and VirB5 proteins and allows contact with the host cell. ii) the transmembrane or "core" channel (VirB7, VirB9 and VirB10) passes through the inner membrane, the periplasm and the outer membrane. This channel allows the transport of the effector. iii) three NTPases provide the energy input necessary for the assembly of the complex as well as the transport of substrates through the pore (VirB4, VirB11 and VirD4) (Figure 3) [START_REF] Lavigne | Type IV secretion system and their effectors: an update[END_REF][START_REF] Christie | Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems[END_REF] .

T4SSs are broadly classified as type IVA with structural components resembling the VirB/D4 complex of Agrobacterium tumefaciens or classified as type IVB, when they resemble the conjugal transfer system of the self-transmissible IncI plasmid, such as the case of Legionella and Coxiella [START_REF] Sexton | Type IVB secretion by intracellular pathogens[END_REF]. Nevertheless, it is important to note that there are important differences between the systems even within the same class. This is nicely illustrated with the example of the type IVA T4SS of Helicobacter pylori. A pathogenicity island called cagPAI encodes for the T4SS known as cagT4SS, responsible for the translocation of the CagA oncoprotein [START_REF] Kaplan-Türköz | Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin[END_REF][START_REF] Kaplan-Türköz | Structure and mode of injection of the oncoprotein CagA of Helicobacter pylori[END_REF].

This island encodes 28 proteins, 12 of which have similarities to the VirB proteins of A. tumefaciens and are essential for CagA translocation. This reveals that T4SS in H. pylori is more complex than its counterpart in A. tumefasciens.

Studies are currently being undertaken to characterize the Brucella's T4SS, which has some intriguing features such as lacking the gene encoding for VirD4, an important ATPase in other systems and encoding for an additional protein (VirB12) with unknown function.

Brucella effectors

In Brucella, the VirB T4SS delivers into the host cells protein effectors that modify cellular functions in order for the bacterium to survive and proliferate [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF]. Mutants lacking virB essential genes cannot survive or replicate in host cells and are attenuated in a mouse model of infection, showing that the virB encoded T4SS plays a major role in Brucella pathogenesis [101].

 Brucella effectors identification:

 Since its discovery 20 years ago, only about twenty effectors have been identified and not all of them have yet been characterized. This contrasts with Legionella, whose T4SS was identified in 1998 [102], with more than 300 effectors characterized [103]. This shows how difficult it has been to identify effectors in Brucella. The Table 2 recapitulates briefly what is known about Brucella VirB-secreted effectors. The majority of effectors in Brucella have been identified using a bioinformatic approach based on the sequenced Brucella genome. This approach aims to identify genes encoding proteins with eukaryotic-like domains, proteinprotein interaction domains, domains present in other bacterial effectors known to be involved in virulence [104], potential horizontally transmitted regions encoding transposases or recombinases adjacent to transfer tRNAs [105] or presence of features similar to known VirB T4SS effectors (eg potential secretion sequences) or distinct GC content [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF].

 Brucella effectors confirmation: Once identified, these putative effectors must be confirmed as translocated in a T4SSdependent manner during infection in the host cell. Several techniques have been successfully used: in particular the use of reporter systems such as CyaA adenylate cyclase or TEM1-βlactamase enzymatic translocation assays [104] [106] and more rarely by directly observing the effector fused to a tag [107]. The position of the N-terminal or C-terminal reporter tag of the putative effector can disturb the translocation of the protein. For example, only the position of the TEM1 in C-terminal of BspB, BspC, BspE showed a translocation while the translocation of BspA and BspF is independent of the position of the tag in N or C-terminal. In addition, several effectors have been shown to be translocated in the host cell but in a manner totally independent of the T4SS (BspG, BspH, BspI, BspK) [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF]. This indicates the need for better characterization of the molecular mechanisms involved in effector translocation via T4SS in Brucella and the caveats associated with the use of reporter translocation systems.

Effector proteins secreted by Brucella T4SSs

The first reported effector protein were VceA and VceC, identified using TEM1-beta lacamase Additionally, BPE005 was shown to induce collagen deposition and matrix metalloproteinase 9 down-modulation via transforming growth factor beta1 in hepatic stellate cells [113].

De Barsy et al. used a high-throughput yeast two-hybrid screen to identify interactions between Brucella proteins and human proteins predicted to be enriched in phagosomes such as RabGTPases. This screening identified RicA (Rab2 Interacting Conserved protein A) as putative effector confirmed by TEM1-β-lactamase reporter assay. RicA preferentially interacts with GDP-bound inactive form of the Rab2. Rab2 is involved in membrane trafficking from the Golgi apparatus to the ER [114]. It has been shown that Rab2 is recruited onto BCVs during infection and is involved in the control of intracellular trafficking of BCVs [START_REF] Fugier | The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication[END_REF]. ΔricA mutant strain loses LAMP1 earlier than the wild type strain. By interacting with GDP-bound Rab2, RicA delays the conversion from eBCV to rBCV [START_REF] De Barsy | Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2[END_REF].

Later on, five more effector protein were described, BspA, BspB, BspC, BspE and BspF [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF]. Some of them were shown to target the host cell secretion pathway, including BspA, BspB and BspF. These three effectors were predicted by bioinformatics approach and confirmed by TEM-1 and CyaA assays. Overexpressed in Hela cells or HEK293T, these effectors inhibit protein secretion. In infection, Brucella also interferes with host protein secretion and this inhibition takes place 8 hours after infection, just before the BCV joins the ER, its replication niche. ΔbspB and ΔbspF mutant strains failed to inhibit protein secretion in infection, whereas ΔbspA mutant strain inhibit protein secretion similar to the wild type strain, suggesting that BspA is not involved in the inhibition of secretion in the context of infection. It seems that the inhibition of secretion in the host by these effectors promotes the formation of the Brucella ER-derived replication niche. Indeed, the triple ΔbspABF mutant leads to a replication defect compared to the wild type strain [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF].

The molecular mechanism by which BspB inhibits protein secretion and its involvement in bacterial replication has been recently studied. BspB interacts with an important complex in the coordination of vesicular trafficking in the host cell secretion pathway: the conserved oligomeric Golgi (COG) complex, that regulates protein secretion and vesicular traffic at the Golgi apparatus. The interaction of BspB with the COG complex, leads to the diversion of COGdependent vesicles towards the BCV to promote rBCV biogenesis. BspB and the COG complex are required for an optimal bacterial replication. Surprisingly, the replication defect of ΔbspB mutant strains is restored by the depletion of Rab2 suggesting that BspB may affect retrograde secretory traffic to redirect COG-dependent Golgi vesicular traffic to the BCV [115].

Although individually the role of these effectors is becoming well established, the effect of the combined actions of these effectors is still unknown. Recently Smith et al have established a link between RicA and BspB during infection. They have shown that BspA and RicA show an epistatic interaction in the replication of Brucella. Although there are some discrepancies in relation to previous reports showing a role of Rab2 and RicA in rBCV biogenesis [80] [84], both effectors seem to be involved in modulation of Golgi apparatus associated vesicular transport dependent on Rab2. The currently proposed model is that RicA by interacting Rab2 inhibits the conversion of eBCV to rBCV, impacting rBCV biogenesis and replication, while BspB counters this effect to promote biogenesis and bacterial replication [116]. It will be important to establish the kinetics of secretion of these effectors. BtpA BtpB were identified together ass T4SS effector protein [117]. BtpA was previously studied by several authors, who showed that BtpA has a TIR domain and is able to interfere with DC maturation [118] [119]. It was also reported that its target was TIRAP [120] [121] and that the protein could inhibit killing by CD8+T cells [122]. BtpA and BtpB through their TIR domain are able to modulate host inflammatory responses during infection specifically inhibiting TLR pathways [117], we will discuss BtpA and BtpB in more detail in the chapter on TIR proteins. Finally the T4SS effector protein of Brucella identified until now is SepA [123]. This protein inhibit the fusion of BCVs with the lysosome. The second part of this thesis manuscript corresponds to my principle PhD project. This work was funded by a young researcher ANR project "NucPath" obtained by Dr Suzana Salcedo. This project aims at characterizing the cellular role of two new effectors in Brucella: NyxA and NyxB. To do this, we are using a multidisciplinary approach combining cell biology, cell imaging, structural biology and biochemistry. We had multiple objectives: to identify the target of these 2 effectors and to understand their role and mode of action in infection; to carry out a structural and functional study in order to define the functional domains involved in the modulation of host cell functions.

For this reason, we are going to present the TIR protein related to the first research project and in a second time introduce the nucleus and the sumoylation corresponding to a compartment and a post-translational modification targeted by the two new effectors identified during my major thesis project.

5. TIR proteins and innate immune system.

Immune system

The immune system is the body's biological defense system. It allows the identification of nonself (proteins, viruses, bacteria, parasitic fungi and other pathogens) and self-defense by controlling the invasion and proliferation of infectious agents. It is composed of a multitude of proteins, cells, tissues and organs forming a dynamic network capable of specifically recognizing and adapting a response to eliminate a wide variety of foreign microorganisms.

The immune system is able to recognize molecular patterns that characterize groups of pathogens with known characteristics, and to establish a rapid immune response directed against these pathogens. It is divided into two parts that differ according to the speed and specificity of the immune response: the innate immune system (non-specific) and the adaptive immune system (specific). The innate immune system is the body's first line of defense against infectious agents while the adaptive immune system acts as a second line of defense.

Nevertheless, there is an strong interconnection between these two immune systems, so the innate immune response stimulates the adaptive response and influences its response [124] [125].

Despite these efficient barriers, some pathogens have developed strategies to circumvent immune defense mechanisms.

In most cases, the innate immunity is very effective and immediately brought into play, preventing most infections from spreading and thus allowing the infectious agent to be eliminated within a few hours of its encounter with the organism [126]. Innate immunity includes several non-specific protective mechanisms such as physical or mechanical barriers.

A good example are epithelial cells (urogenital, bronchial or digestive epithelium). that form a barrier between the external environment where the microorganisms reside and the internal environment which is supposed to be pathogen-free. The pulmonary epithelium for instance, ensures a protective function against external aggressions by the secretion of mucus on which dust, particles and microbes will be trapped. Multiciliated cells, on which several hundred vibrating lashes will beat in a coordinated manner, allow this mucus to be transported to the digestive tract where it will be broken down.

Once infectious agents have penetrated the body, the innate immune system is able to induce an inflammatory reaction using specific receptors. This mechanism involves different cell types such as: phagocytes (macrophages, neutrophils, monocytes and dendritic cells), natural killer (NK) cells and innate lymphoid cells. The innate immune system corresponds to a common defense mechanism in all multicellular animals and is thought to have evolved long before the adaptive immune system [127].

Recognition of microbes and pathogens by the innate immune system

The immune system is able to make "self" and "not self" this distinction by recognizing repeated molecular structural patterns present on the surface of invading microorganisms, whether pathogenic or not.

 PAMPs :

In mammals, the immune response is essentially via effector cells such as macrophages, which are able to detect particular patterns decorating pathogens. These particular patterns have been named by Charles Janeway, PAMPs which stands for Pathogen Associated Molecular

Pattern [124]. Nevertheless, it is important to emphasize that PAMP is not entirely the right term to describe these structures, since they are also present on non-pathogenic microorganisms, especially commensal flora. These molecular signatures are conserved during evolution and are exclusively present on the microorganisms and totally absent from the host cells. These structures are also essential for the survival and proliferation of these microorganisms.

The recognition of the non-self will lead to the production of cytokines such as interleukin (IL)-1, TNFα (Tumor Necrosis Factorα) and IL-6 leading to the inflammatory response and stimulate the adaptive immune response via lymphocytes.

Each group of microorganisms has one or more PAMPs. These unique molecular units present on the surface of the microbes can be: lipopoysaccharide (LPS) present on the outer membrane of Gram-negative bacteria, lipoteichoiic acid and peptidoglycan present on the membrane of Gram-positive bacteria, flagellin in flagella of bacteria, dsRNA and ssRNA in viruses, or mannans on the wall of fungi. These PAMPs will be detected by our body to alert the immune system. How can the immune system recognize these PAMPs?

PAMPs are recognized by specific receptors present on the surface or inside cells of the innate immune system called PRRs (Pattern Recognition Receptors) [128].

 PRRs :

PRRs are receptors expressed in innate immune cells such as macrophages and dendritic cells.

PRRs are also present in non-professional cells, as epithelial cells, endothelial cells and fibroblasts that contribute to the innate immune response. These are present at the plasma membrane or within cells, at the cytosol level but also in different cell compartments such as endosomes. This diversity of cellular localization of these PRRs corresponds to the different cellular compartments in which pathogens can be found. Thus, PRRs are able to recognize pathogens both extracellularly and intracellularly.

Each type of PRR can recognize a multitude of pathogenic species that share a particular molecular motif.

There are 4 main types of PRRs [129] (Figure 4):

-TLRs : Toll-Like Receptors : TLRs were the first family of PRRs to be discovered, they are also the best characterized. TLRs are related to defense against viral, bacterial and fungal infections. We will describe them in more detail later.

-CLRs : C-type Lectin Receptors :

CLRs are a large family of transmembrane receptors present mainly at the plasma membrane level of macrophages and dendritic cells. They are composed of one or more CTLDs (C-type lectin-like domains). They allow the detection of carbohydrate (polysaccharide) motifs contained mainly in fungal walls and leads to an inflammatory response.

-RLRs : RIG-1 like Receptors RLR receptors essentially recognize viral components, mainly viral nucleic acids, and will activate all signaling pathways: NF-κB, MAP-kinases and interferon.

-NLRs : NOD-Like Receptors NLRs are a large family of cytosolic receptors. They are present in cells where they act as sensors of bacterial invasion. They are subdivided into 3 subfamilies: NOD, NALP and NAID. All three subfamilies recruit caspases that cleave a number of cytokines, in particular inflammatory cytokines such as interleukin 1, which is found in the inactive form in the cytoplasm and is thus activated.

Scavenger receptors can also be added to the PRR family because of their involvement in the recognition of PAMPs [130].

Activation of PRRs induces a cascade of intracellular signaling leading to activation and/or modulation of the immune response. This signaling cascade results in the production of antimicrobial peptides, the secretion of pro-inflammatory cytokines and the recruitment of neutrophils and macrophages that will together allow elimination of the invading pathogen.

Hereafter we are going to focus more particularly on the TLR. 134-136] [134]. From an evolutionary point of view, it seems that this TLR recognition system is highly conserved over time.

In mammals, TLR receptors are mainly present on the surface of immune cells such as dendritic cells, macrophages, polynuclear cells, B and T lymphocytes. They can also be found in a large number of cells having contact with the external environment such as skin cells, epithelial cells or intestinal cells.

 TLR structure:  TLRs are type I membrane receptors in which three domains can be distinguished (Figure5):

-An extracellular N-terminal domain, which allows ligand recognition; this domain corresponds to a leucine-rich repeat (LRR).

-A transmembrane domain, -An intracellular C-terminal domain that allows signal transduction, thanks to a specialized domain called TIR, for Toll/interleukin-1 receptor homology.

Extracellular domain :

The extracellular domain of TLRs is composed of approximately 800 amino acids in the form of leucine-rich repeats called Leucin-Rich Repeats or LRRs. This extracellular domain is involved in pathogen recognition and plays a key role in the initiation of TLR signaling [135].

The distribution and number of leucine-rich repeat units is specific to each TLR, which gives them their specificity in pathogen recognition [136,137]. It is composed of a beta-sheets and an alpha helix that forms a horseshoe structure, the beta-sheets are present on the concave side of the structure (Figure 6).

Intracellular domain :

The intracellular TIR domain of TLRs, consisting of 150 to 200 aa, is similar to that of the IL-1 These proteins allow, once the TLRs are activated, the transduction of the signal. Proteins containing TIR domains share 20-30% sequence identity. From a structural point of view, the TIR domains adopt approximately the same three-dimensional structure, with 5 parallel beta sheets surrounded on both sides by 5 alpha helixes. The protein structure is more stable but also more conserved than the sequence during evolution. When comparing the structural alignment of the TIR domain of TLR2 (figure 7C) with that of the MyD88 TIR domain (Figure 7B) and calculating the Root-Mean-Square Deviation (RMSD) of each of the alpha carbons, an RMSD of 2.431Å is obtained, which allows us to consider that the two structures are relatively close to each other (Figure 7D). This is why we can say that the TIR domains are domains conserved during evolution.  TLR and their Ligands : 

To date there are 10 different TLRs in humans (TLR1 to TLR10) and 12 TLRs have been identified in mice (TLR1 to TLR13), TLR10 is not present in mice [144]. Two different TLR groups can be distinguished according to their location in the cell. The TLRs present on the cell surface TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10, and the TLRs present in the cell at the endosomes:

TLR3, TLR7, TLR8, TLR9 [145] (Figure 8).

Each TLR is specific to a component of the microbes (Table 2). For example TLR2 recognizes bacterial lipoglycans, peptidoglycans TLR3,7 and 8 recognizes viral nucleic acids, TLR4

recognizes LPS, TLR5 recognizes flagellin, TLR 9 recognizes unmethylated oligonucleotide motifs. Once PAMP has been detected, TLR dimerize into either a homodimer or a heterodimer [146].

TLRs will activate an intracellular signaling cascade which is initiated by recruiting via TIR-TIR interactions between the TIR domain of the TLRs and the TIR domains adaptor proteins. This interaction allows the recruitment of IL-1R-associated protein kinases (IRAK) 1, 2, 4, M, TAB2

and TNF receptor-associated factor 6 (TRAF6). This signaling cascade leads to the translocation of transcription factors in the cell nucleus such as NF-B [147], members of the IRF family (IRF3 8). These transcription factors allow the activation of genes and thus the production of inflammatory cytokines such as TNF alpha, IL-6, IL-1beta and IL-6 but also the production of chemokines (CXCL8 and CXCL10) and type I interferon (IFN)

[150].

TLRs induce a specific response to their ligand. This specificity is mediated by the adaptor proteins involved in signal transduction. ). TRAF6 will thus be able to ubiquitinate itself. Ubiquitinylate TRAF6 will be recognized by TAB2 (TAK1 binding protein) and TAB3, leading to the activation of the TAK1 complex (transforming growth factor [TGF] -activated kinase 1). The activated TAK1 complex generates a phosphorylation cascade of the different members of the MAPK (Mitogenactivated protein kinase) pathway but also the induction of IB (inhibitor of NF-B )

degradation via activation of the IK (IB kinase) complex, leading to the release of NF-B and its nuclear translocation [154]. Activation of the MAPK pathways and the NF-B transcription factor allows the expression of inflammatory cytokines.

 TRIF-dependent signaling pathway 

The second TLR pathway induced following pathogen recognition is independent of Myd88 and is associated with TLR3 and TLR4[155]. This alternative pathway is dependent on TRIF, which is recruited directly at the TIR domain of TLR3 or indirectly via TRAM for TLR4.

Subsequently TRIF interacts with TRAF6 and RIP1 (Receptor-interacting protein 1) to activate the MAPK and NF-B pathways in the same way as the MyD88-dependent signalling pathway.

However, TRIF also collaborates with TRAF3 which activates IKK??inhibitor of NF-κB kinase ? and TBK1 (TRAF family member-associated NF-B activator-binding kinase 1), both of which are responsible for phosphorylation and nuclear translocation of IRF3 and IRF7. This pathway thus allows the expression of type I IFNs and mainly IFN- [156].

Microbial targeting of the TLR signaling pathway

Over the course of evolution, bacteria have developed different strategies to subvert immune responses from the host cell, including hijacking signaling pathways, modifying their PAMPs, and activation of others receptors that dampen the effect of TLR activation (Figure 9) [157].

Some pathogenic bacteria have evolved by modifying their PAMPs so that they are less Brucella is also known to modify its PAMPs to escape the immune system or limit the host's immune response. Firstly, Brucella does not express pili, fimbriae or capsules which are recognized by the immune system. Furthermore, Brucella melitensis seems to produce a nonfunctional flagellum which limits its recognition by the Therefore, in the introduction I have decided to briefly present the different structures of the nucleus and their functions, the process of SUMOylation and finish with a more detailed description of the functions of SENP3.

The nucleus :

The nucleus can be considered as the heart of the cell. It was the first cell compartment to be discovered in 1833 by Robert Brown. It contains the vast majority of the cell's genetic material, which is present as compacted DNA in the 23 pairs of chromosomes, with the rest of the genetic material found in the mitochondria. The chromosomes are present in defined regions of the nucleus in interphase [191]. The nucleus is highly organized and dynamic. It protects the genetic material and controls the physiology of the cell, particularly during cell division. It also performs major functions: DNA repair, transcription, RNA splicing, DNA replication, ribosome assembly, chromatin modifications, gene regulation and expression.

Only the cells of higher organisms (eukaryotes) have a nucleus, with the exception of red blood cells. The nucleus is the largest organelle of the cell, separated from the cytosol by a nuclear envelope corresponding to a double lipid membrane. The nuclear envelope has nuclear pores allowing the transport of biomolecules in both directions between the cytoplasm and the nucleus. The nucleus represents about 10% of the total volume of the cell with a diameter of about 10µm, however this value varies greatly depending on the cell type and the stage of the cell cycle. In Hela cells the nucleus occupies 8-21% of the total cell volume. The protein atlas website (https://www.proteinatlas.org/humanproteome/cell) provides information on the expression and spatio-temporal distribution of proteins in human cells. They have been able

to establish experimentally that 33% of all human proteins (i.e. 6523 proteins) are located in the nucleus. The presence of such a large number of proteins in a relatively small space underlines the need for structural organization in the nucleus.

Architecture of the nucleus :

Much is known regarding nuclear molecular mechanisms and processes, however it is only recently through molecular biology, biochemistry and cell biology techniques and especially through technological advances in the field of imaging using increasingly powerful microscopes that the architecture of the nucleus and its three-dimensional organization is beginning to be elucidated. In the 1980s, the use of fluorescent probes in microscopy made it possible to revive the study of the architecture of the nucleus by observing nuclear proteins of chemically fixed cells [192]. This made it possible to highlight the presence of different structures within the cell with distinct and highly dynamic morphologies. To date, several nuclear sub-structures have been identified, also called nuclear bodies.

The different nuclear compartments: nuclear bodies

In a similar way to cytosol, where a compartmentalization of the different organelles can be observed according to different metabolic processes (i.e. mitochondria, Golgi apparatus, endoplasmic reticulum...), the cell nucleus presents different sub-structures [192] [193].

However, unlike the cytosol, the different nuclear sub-structures are not separated from their surroundings by a membrane. This compartmentalization facilitates metabolic processes, According to 3[198] integrity is ensured by protein-protein and protein-RNA interactions, yet the mechanisms of formation of these structures are still poorly understood [194].

Numerous nuclear bodies have been described and characterized: nucleoli, nuclear speckles, clastosome, PML bodies, Cajal bodies, Gems bodies, paraspeckles, histone bodies and stress bodies [195] (figure10). Hereafter we will discuss only the nucleoli and PML nuclear bodies.

The nucleolus: an assembly platform

The nucleoli form the largest and most dense structures of the nucleus, first observed by microscopy at the end of the 18th century (Fontana, 1781). Fontana characterized it as an "oviform body with a spot in the middle". Most mammalian cells contain 1 to 5 nucleoli with a diameter of 0.5 to 5 µm. [198] They are mainly known for their role in the production of ribosomal RNA (rRNA) and in the assembly of ribosomes. In addition, the nucleoli are involved of the cell's metabolic activity. Indeed, rRNA synthesis is linked to cell growth and activity [196]. Dividing cells generally have large nucleoli to ensure a high level of ribosome biogenesis for sustained protein synthesis.

There is therefore a positive correlation between rRNA synthesis and nucleolar size.

Conversely, cell cycle arrest leads to a reduction in nucleolar size [197]. The nucleolus adopts a structure divided into three zones: a fibrillar centre, a dense fibrillar component and a granular component (figure 11) [198].

The Fibrillary Centre (FC) :

It is a rounded area of the nucleoli not very dense to electrons, located in the centre in quiescent cells, more in periphery and less regular in the most active cells. This zone contains the promoter regions of the rDNA genes and is the site of initiation of rRNA transcription.

The dense fibrillar component (DFC) :

DFC is very dense to electrons, which is adjacent to the fibrillar centre. Limited to a small proportion of the nucleolus in quiescent cells, it becomes more important in proliferating cells and forms filaments called nucleonemes. FC and DFC participate in the transcription of rRNA.

The rRNA thus produced is called precursor or pre-rRNA. The pre-rRNA then undergoes several stages of maturation within DFC.

The granular component (GC) :

GC is an area larger than CF and CFD located at the periphery of the nucleoli. The granular aspect of GC is due to the large number of pre-ribosomal particles being assembled or stored.

Although it is clear that nucleoli play a role in cell growth via ribosome biogenesis, the notion of nucleoli multifunctionality is emerging. Indeed, proteomic analysis reveals that about 30% of nucleoli proteins are involved in ribosome biogenesis, suggesting that nucleolus are likely to be involved in various other cellular processes [199]. Nucleoli are believed to be involved in the maturation and export of certain mRNAs and tRNAs, DNA replication and repair, cell cycle progression, proliferation and apoptosis [200] [201]. Indeed, nucleoli are sensitive to a variety of stresses and trigger a rapid response by regulating the induction of p53, a key transcription factor regulating multiple cellular processes including proliferation and death [196].

 The production of ribosomes by nucleoli :

Ribosomes :

Ribosomes are made up of proteins and ribosomal RNA (rRNA) to form ribonucleoprotein complexes. They are found in both eukaryotic and prokaryotic cells. These structures are relatively well preserved during evolution, mediating protein synthesis proteins by decoding the information contained in messenger RNA (mRNA). Ribosomes are the main effectors of translation and therefore are intimately linked to the capacity of cells to grow and divide [202].

The ribosomes are formed of two subunits: a small subunit that allows binding to the messenger RNA and a large subunit whose function is to carry out the polymerization of the amino acid residues to form the corresponding protein. These sub-units are designated according to the sedimentation coefficient expressed in Svedberg (S) units.

In eukaryotes, the large subunit has a sedimentation coefficient of 60S with a molar mass of 2.9 MDa. It consists of three ribosomal RNAs: 28S rRNA, 5.8S rRNA and 5S rRNA with 49 ribosomal proteins. The small subunit has a sedimentation coefficient of 40S with a molecular weight of 1.4 MDa. This sub-unit is composed of a single ribosomal RNA, the 18S rRNA associated with 33 ribosomal proteins. The rRNA is the major constituent of ribosomes, it represents 60% of the total mass of ribosomes.

Ribosome biogenesis

Ribosome biogenesis in eukaryotes is a complex process of transcription, maturation, folding and post-translational modification of rRNAs. This process takes place in several stages with different locations in the cell: it starts in the nucleoli, then during their maturation the preribosomal particles join the nucleoplasm and cross the nuclear pore to reach the cytosol where they are finally assembled [203]. It involves more than 200 partners (protein and small nucleolar RNAs (snoRNAs)) who coordinate the different stages of maturation, pre-rRNA cleavage, post-translational modifications of ribosomal proteins and the export of sub-units into the cytosol [206][207][208][209]. Although a large number of proteins have been identified as ribosome-associated by co-immunoprecipitation or mass spectrometry, the molecular functions of a large number of proteins involved in ribosome biogenesis are still unknown. This process is extremely energy intensive for the cell. It is estimated that 80% of the cell's energy reserves are required for ribosome biogenesis [204].

Ribosome biogenesis is globally well conserved between eukaryotic species. The model is best established in yeast (Saccharomyces cerevisiae) and is often extrapolated to mammals and humans. Nevertheless, many differences can be observed in the structure, factors involved and maturation stages of pre-rRNAs [211][212][213]. In mammals, it involves the three RNA polymerases (I, II, III). RNA polymerase I allows the transcription of 28S, 18s and 5.8S rRNAs.

RNA polymerase II synthesizes the mRNA of ribosomal proteins. RNA polymerase III synthesizes rRNA5S.

Biogenesis starts in the fibrillar center of the nucleoli, where RNA polymerase I allows the transcription of 47S RNA precursors. The latter contains the sequences of the 5.8S, 18S and 28S rRNAs surrounded by external transcribed spacers at the 5' and 3' ends (5'-ETS and 3'-ETS), and are separated from each other by internal transcribed spacers (ITS-1 and ITS2). This 47S transcript will undergo a succession of endonucleolytic cleavage and exonucleolytic digestions, these processing and cleavage steps performed by several ribosomal proteins that participate in the ribosome structure, different assembly factors as well as non-coding RNA species [205]. The assembly factors assist the formation of the maturation of the subunits but will not be present in the final architecture. Thus, the 47S rRNA precursor will first lose the 3' ETS then the 5' ETS to form the 45S rRNA precursor then the 41S rRNA precursor. At this stage the pre-rRNA processing will split in two pathways. The first will lead to the pre-rRNA18S which will be incorporated, together with ribosomal proteins, in the pre-ribosomal 40S subunit [206].

The second pathway will undergo additional cleavage steps to obtain the 5.8S and 28S rRNA.

At the same time, the 5S rRNA that follows a different assembly pathway, will be transcribed by RNA polymerase III at the periphery of the nucleoli. Subsequently, it will be recognized by two ribosomal proteins uL18 (Rpl5) and uL5 (Rpl11). It will then join the nucleoli where it will associate with 28S and 5.8S rRNA and ribosomal proteins to form the 60S pre-ribosomal subunit. These steps of cleavage and maturation of the spacer regions are performed in the nucleoli. These pre-ribosomal particles (40S and 60S) undergo several steps of cleavage and maturation in nucleoplasm, and are exported to the cytoplasm to form the mature 40S and 60S sub-units and subsequently form the 80S ribosomal particle, capable of mRNA translation (figure 12) [207].

The PML nuclear bodies:

PML nuclear bodies are structures composed of multi-protein complexes defined by the presence of the PML (promyelocytic leukaemia) protein itself. These bodies have a punctate appearance in the cell nucleus that vary in number from 5 to 20 per interphase nucleus, with a diameter of 0.5 µm. The main organizing protein of these nuclear bodies is the PML protein, whose modification by SUMO is necessary for its stability and PML-NB formation [216,217].

Indeed, only the form of PML conjugated to SUMO is able to properly aggregate and induce the formation of nuclear bodies. PMLs are regulated by the desumoylases of the SENP family (SENP1, SENP3, SENP5 and SENP6) [218][219][220]. Nevertheless, the mechanisms of action of SENPs on PML are still poorly understood. In the presence of arsenic, the SUMOylation of PML is exacerbated, and an increase in the size of PML nuclear bodies is found [208].

PML nuclear bodies recruit different proteins, the only common characteristic of which is their ability to be sumoylated [209]. These include the proteins PML, SP100, BLM, Daax, LEF1, p53, CBP... [210]. PML nuclear bodies are proposed to be reservoirs of regulators involved in the control of many cellular processes such as stress response, apoptosis or response to DNA damage. They constitute hubs of further post-translational modifications, including ubiquitination. For the cell, PML nuclear bodies are likely to be a repository of nuclear factors and a specific site for modification and assembly of transcriptional factors [211] [212]. It is also interesting to note that the depletion of PML in cells leads to an acceleration of cell proliferation [213].

PMLs have been studied in particular because of the alteration of PML-NB in pathological conditions such as promyelocytic leukemia, viral infections or various cellular stresses [214] [215] [216]. Shigella infection was also shown to impact PML nuclear bodies by increasing the number of PMLs in infected cells [217], following decrease in the levels of host sumoylation.

Recently, PML was identified as being targeted by the pore-forming toxin (LLO) of Listeria monocytogenes. LLO triggers a de-SUMOylation of PML, which leads to a de-structuring of the PML nuclear body and triggers an antibacterial response via activation of immune response genes and cytokine secretion [218]. Further studies with other intracellular bacterial pathogens need to be undertaken to determine whether PMLs play a general antimicrobial control mechanism.

SUMOYLATION :

The cell can mediate the dynamic regulation and biochemical functioning of its proteins through post-translational modifications (PTM), that correspond to the attachment of a group to a target protein, which will modify its properties and consequently its functions. These include: glycosylation, acylation, phosphorylation, acetylation, methylation, ubiquitination... Sumoylation is also a post-translational modification of target proteins. It is an extremely dynamic and reversible process. SUMO was initially identified in 1995 in Saccharomyces cerevisiae with the smt3 gene, a homologue of SUMO1, following genetic screening to identify suppressors of a mutation in MIF2, a protein associated with the centromere [219]. In 1996, three teams simultaneously discovered SUMO1, through yeast two-hybrid experiments. In these studies, SUMO1 was shown to interact with PML [220], Rad51 and Rad52 involved in DNA repair [221] and Fas protein playing a role in apoptosis [222], already suggesting a high diversity of mechanisms in which this PTM is involved.

In recent years many more SUMO substrates have been identified, underlining the importance of this SUMO modification within the eukaryotic cell [223] [224].

SUMOylation consists of the addition of a polypeptide called SUMO (Small Ubiquitin-like MOdifier) of about 10kDa which shares 20% identity with ubiquitin. Nevertheless, the threedimensional structure of these two proteins is relatively close (Figure 13). The addition of this SUMO polypeptide is achieved by forming an isopeptide bond with the -amino group of the lysine acceptor residues of the target protein. SUMOylation is involved in a multitude of cellular functions such as transport between the nucleus and the cytosol, transcriptional regulation, apoptosis, protein stability, immune response, DNA repair, stress response, cell cycle progression, cell proliferation and differentiation [235,237,238]. For example, mice in which expression of Ubc9, a key enzyme in the SUMOylation pathway, is turned off, die in the embryonic stage [239,240]. This underlines the importance of SUMOylation in cell proliferation and differentiation.

Initially, the scientific community thought that sumoylation occurred only in nuclear or perinuclear compartments [225], but it has become clear that it also regulates cytoplasmic and plasma membrane proteins [226]. SUMO proteins are present in all eukaryotes. Some organisms have only one SUMO gene such as yeasts (SMT3), Caenorhabditis elegans (SMO-1)

and Drosophila melanogaster (smt3) [242][243][244] , whereas plants and vertebrates have several SUMO genes. In humans, 5 paralogues of SUMO have been identified: SUMO1, SUMO2, SUMO3, SUMO4 and more recently SUMO5 [227] [246,247]. SUMOs are formed by about 100 amino acid residues. SUMO2 and SUMO3 are very strongly similar and share 97% identity, only three amino acids differ. As a result, it is very complicated to differentiate between them and they are often referred to SUMO2/3. SUMO2/3 shares 47% identity with SUMO1 and 83% identity with SUMO4 [228] and 46% identity with SUMO5 (figure 14). Proteomic analyses have shown that 2% of the mammalian proteome is SUMOylated underlining the importance of this PTM [229]. SUMO1 and SUMO2/3 perform very distinct functions in the cell, as they are conjugated to different target proteins in vivo [250][251][252]. The role of SUMO4 is more obscure, currently scientists are not sure whether it is in the mature in vivo form needed for conjugation [245,253]. Results suggest that SUMO4 is involved in the pathogenesis of type I diabetes [228]. The youngest member of the SUMO5 family seems to play a major role in the regulation of the nuclear PML bodies [230]. It should be noted that SUMO1, SUMO2/3 have been studied in more detail than the SUMO4 and SUMO5 paralogues.

To date, the mechanisms and consequences of this specificity of conjugation of the SUMO paralogues to their substrate are not clearly established and many questions remain unanswered. Like ubiquitination, proteins can be modified with the addition of a single SUMO molecule (monoSUMOylation) or with several chains of SUMO molecules (polySUMOylation) [231]. Nevertheless, only SUMO2/3 and SUMO4 are capable of producing polySUMO chains, due to the presence of a lysine 11 present in a KxD/E consensus sequence in SUMO2/3 and SUMO4 to which SUMO is added, but absent in SUMO1 and SUMO5 (Figure 14) [255,256]. On the contrary, lysine 18 can be observed on SUMO5, which could be SUMOylated, but to date no study has reported a polySUMOylation of SUMO5.

The enzymatic mechanism of SUMOylation :

The different paralogues of SUMO are conjugated by covalent binding to the target protein at the lysine level generally present in KxD/E consensus motif (in which  is a branched aliphatic amino acid, K corresponds to lysine, x any amino acid, D corresponds to aspartic acid E corresponds to glutamic acid) [257,258]. It should be noted, however, that the literature reports an increasing number of sumoylated proteins on lysines not belonging to a consensus sumoylation site. In addition, many proteins with this motif are not sumoylated.

 Maturation of the precursor: 

The various forms of SUMO are expressed as inactive precursors that require cleavage of the C-terminal part of the peptide to expose a di-glycine motif essential for conjugation. It is at the carboxy-terminal glycine doublet (GG) that the binding with the lysine of the target protein will take place. This cleavage is achieved by the hydrolase activity of specific enzymes of the SENPs family (SUMO/Sentrin-specific protease). Similar to ubiquitination, SUMOylation consists of a cascade of enzymatic steps involving three enzymes: SUMO activating enzyme (E1), SUMO conjugating enzyme (E2) and ligase (E3) (Figure 15).

 -E1 the activating enzyme : 

Unlike ubiquitination, the E1 enzyme is not monomeric but heterodimeric. This heterodimeric E1 enzyme consists of two subunits: a small SAE1 subunit and a large SAE2 subunit (SUMO Activating Enzyme 1 and 2). This activation is ATP dependent. Initially E1 forms a high-potential energy connection between the C-terminal end of SUMO and SAE2 using ATP. Then, by releasing the AMP, the adenylated SUMO intermediate uses this binding energy to form a covalent thioester-type bond between the carboxyl group at the C-terminal end of SUMO and the sulphide group of the C173 residue of the SAE2 sub-unit [232].

 -E2 conjugating enzyme: 

In contrast to ubiquitinilation, SUMOylation uses exclusively the conjugating enzyme E2: UBC9 (SUMO ubiquitin-conjugating enzyme 9). UBC9 is highly conserved in eukaryotes, it has 56% amino acid sequence identity between the human UBC9 sequence and its orthologue in S. cerevisiae [233]. By a transesterification reaction, "activated" SUMO is transferred from SAE2 to the cysteine C93 residue of the conjugating enzyme Ubc9, again forming a covalent thioesther bond. Ubc9 serves as the donor of SUMO. Ubc9 will then create an isopeptide bond between the double glycine of SUMO and the lysine of the substrate  -E3 SUMO ligase :  It is important to note that in vitro tests show that the activating enzyme E1 and the conjugating enzyme E2 are sufficient for SUMOylation of the substrate [261][262][263].

Nevertheless, the conjugation process can be assisted by E3 ligases which promote the interaction of the E2 enzyme with the substrate or act by positioning SUMO in a conformation that facilitates its transfer to the target lysine residue [234]. Thus, the conjugation of SUMO is generally increased in the presence of E3 ligases in vivo and in vitro [265,266]. The three main types of E3 SUMO ligases: the PIAS family (Protein Inhibitor of Activated STAT), the nucleoporin RanBP2 (Ran Binding Protein2) and the Polycombe PC2 complex, interact with Ubc9 and the substrate [236,267]. SUMOylation: firstly, they allow the maturation of newly translated SUMO into its active form thanks to their hydrolase activity, which is necessary for conjugation, and secondly, they allow the removal of SUMO from their substrate by an isopeptidase activity.

DeSUMOylation seems to be essentially exercised by SENPs, in fact cells lacking members of the SENPs family present a large accumulation of SUMOylated proteins. Conversely, cells lacking DESI1, DESI2 or ULSPL1 do not present an alteration in the total SUMOylated protein profile. This suggests the major role of the SENPs family in the overall status of cell SUMOylation. However, the study of this post-translational modification is complicated by the fact that this event is highly dynamic. Only a small fraction of substrate is SUMOylated at any given time. It therefore seems that there is a SUMOylation/de-SUMOylation equilibrium for a specific substrate and that this equilibrium is finely regulated. To date many questions, remain unanswered about the understanding of the cellular signals that trigger de-SUMOylation and how SENPs are regulated under physiological as well as pathological conditions.

ThE SUMO Interacting Motif (SIM: SUMO Interacting Motif):

Interestingly, on some proteins, a SUMO interacting motif (SIM) has been identified to interact non-covalently with SUMOylated proteins [238]. The SIM motif corresponds to a short sequence not exceeding 10 amino acid residues, may contain phosphorylated amino acids, and interacts with a specific groove present on the surface of SUMO [239]. To date, two important characteristics of SIM motifs have emerged: Presence of a core formed by 3-4 hydrophobic residues (generally valine or isoleucine) and the presence of a close acidic region such as the side chains of glutamic or aspartic acids or phosphorylated residues such as serine or threonine [272,274].

A study on the PML protein showed that it has this non-covalent binding motif, which is involved in the formation of PML nuclear bodies [240].

Similar to the consensus site of SUMOylation KxD/E, not all SUMO binding motifs are functional, due to poor exposure of the SIM motif, or because these SIM motifs are masked by interaction with other proteins.

Consequences of SUMOylation

Modification by SUMO can have several consequences on the target protein, and it can change its localization, activity or stability. SUMO can compete with other PTMs which modify the function of the protein, or SUMO can modify directly the conformation of the target protein.

SUMO can also modulate the interaction with other proteins, by preventing protein-protein interactions or conversely by promoting new interactions by exposing a new binding domain or directly binding to another protein with a SIM (figure 16) [241].  Bacterial effectors can mimic host deSUMOylases.  This is the case for Yersinia outer membrane protein J (YopJ), the first bacterial protein identified to modulate SUMOylation. YopJ is a cysteine protease that mimics the isopeptide activity of SENPs. It will lead to the deconjugation of SUMO from their substrate and thus to a decrease in the overall level of SUMOylated proteins [283,284].

Similarly, XopD is an effector secreted by the T3SS of a plant pathogen, Xanthomonas euvesicatoria. XopD has an isopeptidase activity that allows it to deconjugate SUMO from the transcription factor SIERF4. Thus, the bacterium suppresses the ethylene response allowing it to escape the plant immune system and promote its proliferation [285,286] .

Klebsiella pneumoniae is a major multidrug-resistant pathogen causing nosocomial infections worldwide. Recently, it has been shown that K. pneumoniae decreases the level of SUMOconjugated proteins in epithelial cells but also in macrophages. In epithelial cells, this decrease is not dependent on an alteration of the E1 or E2 enzymes. The authors have shown that SENP2 deSUMOylase is involved in the decrease of SUMO-conjugated proteins. K. pneumoniae triggers the delocalization of SENP2 from the nucleus to the cytosol. The bacterium leads to the deNEDDylation of E3 ubiquitin ligase which results in a defect of ubiquitylation of SENP2 by this ligase and thus prevents the degradation of SENP2 by the proteasome. On the other hand, in macrophages, the decrease in SUMOylation is mediated by type I interferon in a TLR4-dependent manner. The type interferon stimulates the transcription of miRNA let-7 which prevents SUMOylation. This decrease in the SUMOylation status of epithelial cells and macrophages allows intracellular survival of Klebsiella pneumoniae and limitation of the inflammatory response [247].

 Bacteria using the host's machinery to SUMOylate their own effectors  This has been observed in Ehrlicia chaffeensis and Anaplasma phagocytophilum [288,289].

Thus the TRP120 and AmpA effectors belonging respectively to Ehrlicia chaffeensis and Anaplasma phagocytophilum are secreted in the host cell cytosol and localize to the membrane of the pathogen-containing vacuole. TRP120 and AmpA are sumoylated during the infection. SUMOylation of TRP120 allows it to interact with host proteins and thus promote infection [248]. Inhibition of the SUMO pathway significantly decreases the interaction of TRP120 with its protein targets, resulting in decreased intracellular survival of Ehrlicia chaffeensis. However, the molecular mechanisms and repercussions of this modification are not yet established. Shigella flexneri also uses the host machinery for SUMOylate OspF, an effector secreted during infection. The sumoylation of OspF allows it to be translocated into the nucleus of the infected cell where it can modulate proinflammatory cytokine expression [249].

Thus, by interfering with the SUMOylation of the host cell, bacterial pathogens can modulate the activity of proteins in order to promote the replication and dissemination of the bacterium in its host. It seems obvious today that the understanding of these interactions between the host and the pathogen modulating sumoylation is more than necessary and could be used in the future for the development of new therapeutic strategies

The SENP family

SENPs are cysteine proteases, discovered in the early 2000s [237]. They have a C-terminally conserved catalytic domain of about 200 amino acid residues and exhibit a characteristic papain-like folding. The cysteine residue of the active site is present at a catalytic triad (histidine, aspartic acid and cysteine). The N-terminal domain is different among members of the SENP family (Figure 17). This domain is variable in length and plays a major role in their regulatory function and location. It is often subject to post-translational modifications such as phosphorylation or ubiquitination, allowing regulation for partner recruitment or protein stability. We also find SIM motifs allowing them to target their substrates. In humans, there are 6 isoforms of SENPs: SENP1, SENP2, SENP3, SENP5, SENP6 and SENP7.

The 6 members of this family can be divided into three subfamilies according to their sequence identity within their catalytic domain, substrate specificity and cell location [223].

Indeed, they present 20 to 60% identity in their catalytic domain [250]. The first sub-family is composed by SENP1 and SENP2. They do not present any specificity for SUMO, they act as well on SUMO1 as on SUMO2/3. The second sub-family is made up of SENP3 and SENP5 and the last sub-family is made up of SENP6 and SENP7. With the exception of SENP1 and SENP2, all other isoforms of SENP act preferentially on SUMO2/3. Each member of the SENP family has a characteristic distribution, allowing them to target their substrate more specifically. SENPs are located almost exclusively in different nuclear substructures (Figure 18). For example, SENP3 and SENP5 are mainly present in the nucleoli thanks to Nucleolar Localization Site (NoLS) where they are involved in ribosome biogenesis steps [218,[292][293][294]. They can also be found in the nucleoplasm and cytoplasm. Surprisingly, SENP5 moves to mitochondria during mitosis [251].

SENPs key regulators of macromolecular assembly occurring in the nucleus. They are thus indispensable for the formation of PML nuclear bodies and the assembly of pre-ribosomes.

They also play a role in chromatin remodeling and control of gene expression. SENPs are also involved in cell cycle progression, inflammatory signalling and the adaptive immune response.

In the framework of this thesis project we will focus on the role of SENP3.

SENP3

SENP3 is a cysteine protease. This protein is composed of 574 amino acid residues with a molecular weight of 65 kDA. Its catalytic domain is located at the C-terminus of the protein and presents a catalytic triad of cysteine, histidine and aspartic acid. Cysteine 532 is the key residue that enables it to attack the peptide bond between the glycine terminal residue of SUMO and the lysine residue of the substrate. The mutation of Cys532 to alanine leads to a total loss of its isopeptidase activity. SENP3 acts preferentially on substrates sumoylated by SUMO2/3 and has a very limited activity on SUMO1. Depletion of SENP3 by siRNA leads to a significant increase in SUMOylated substrate by SUMO2/3 in cells [293,296,297]. The result of this stabilization under oxidative stress is a redistribution of SENP3 from the nucleoli to the nucleoplasm where it will accumulate and participate in the de-SUMOylation of a large number of proteins. Notably, nucleoplasmic SENP3 will deconjugate SUMO2/3 from p300, a co-activator of HIF-1, a transcription factor sensitive to redox. This deSUMOylation by SENP3 will lead to a transactivation of HIF-1 [257]. A few years later the same team showed that SENP3 is induced under mild oxidative stress but under high oxidative stress its catalytic activity is inhibited due to the oxidation of catalytic cysteine532, preventing the transcriptional activity of HIF-1 [259].

Under oxidative stress, the delocalization of SENP3 nucleoli leads to accumulation within PMLs and thus deconjugation of PML from SUMO2/3. As a consequence, a decrease in the number of PML nuclear bodies, which requires the sumoylation of PML for their formation is observed under oxidative stress.

SENP3 as a modulator of gene expression

Studies have shown that deSUMOylation mediated by the SENPs family has a role in bone metabolism. SENP3 is found associated with the MLL1/MLL2 histone methyltransferase complex and catalyzes the SUMO deconjugation of RbBP5 required for the activation of Hox gene. SENP3 promotes ostogenesis by deSUMOylating RbBP5, which activates the expression of HOX genes necessary for cell specialization and determine pathway such as osteogenesis [250]. Recently a study showed that in bone marrow-derived monocytes, SENP3 activity is decreased during osteoclastogenesis promoting osteoclast differentiation. The authors showed that SENP3 negatively regulates osteoclast differentiation by deconjugating SUMO2/3 present on IRF8 lysine K310. This suggests a potential role for SENP3 as a therapeutic target in diseases related to bone loss [261].

SENP3 is also phosphorylated by the CDK1 protein kinase before entering mitosis and is dephosphorylated by the protein phosphatase-1 (PP1) at the exit of mitosis. The phosphorylation of SENP3 negatively regulates its activity, so that it can no longer desumoylate chromosome-associated proteins such as Topoisomerase ll α (TopoII α). The expression of a mutant of SENP3 that cannot be phosphorylated decreases the SUMOylation of TopoII α, leading to abnormal chromosome segregation, abnormal mitotic cell cycle and tumerogenesis [262]. These data show that SENP3 phosphorylation plays a crucial role in the regulation of chromosome stability in mitosis. Subsequently the same team showed that in response to DNA damage, p53 is activated and will suppress the phosphorylation of SENP3.

SENP3 and immune response :

It is well established that protein SUMOylation plays a role in the innate immune response.

ROS are produced in abundance during macrophage activation and are necessary for inflammatory signaling by TLR4. As we have seen above, SENP3 is sensitive to ROS. SENP3 deficient cells markedly deregulate the activation of TLR4 inflammatory signaling and the production of pro-inflammatory cytokine in LPS-stimulated macrophages. SENP3 potentiates LPS-induced TLR4 signaling via MKK7 desumoylation resulting in increased JNK phosphorylation and downstream events. This suggests that SENP3 may be the link between redox regulation and innate immune response [263].

Finally, NLRP3 (NOD-like receptor family, pyrin domain containing3) which is an inflammasome activating protein undergoes SUMOylation, essential for the regulation if its inflammatory activity [264]. Subsequently, it was shown that SENP3-mediated deSUMOylation of NLRP3 orchestrates the activation of the inflammasome [265].

SENP3 as a regulator of macromolecular assemblies in the nucleus : ribosome biogenesis

As mentioned above, SENP3 is mainly localized in nucleoli, a major site of ribosome biogenesis due to the presence of a nucleolar localization sequence (NoLS). However, authors have also shown that this nucleolar localization is also dependent on the serine/threonine kinase activity of mTOR. Indeed, when HeLa cells were treated with mTOR inhibitors (Ku-0063794, Rapamycin), SENP3 was no longer able to localize into the nucleoli, revealing that phosphorylation of several N-terminal SENP3 serines and threonines (S25, S139, S141, T142, S143, T145) by mTOR was required for its localization. This delocalization of SENP3 is also observable in case of amino acid starvation, consistent with the fact that during starvation mTOR is no longer activated [266].

When SENP3 is phosphorylated, it interacts with its major partner nucleophosmin (NPM1/B23) which will allow SENP3 to be retained in the nucleolus In addition, a large protein complex associated with SENP3 has been identified, it is composed of PELP1, TEX10 and WDR18. PELP1 is found in the GC region of the nucleolus, associated with the precursor forms of 28S rRNA, and it interacts with LAS1L [270], a 28S rRNA maturation factor. When PELP1 is sumoylated it is localized in the nucleoplasm while when it is not conjugated to SUMO it is found in the nucleolus. Thus, SENP3 will regulate the nuclear distribution of this complex. The authors suggested that SENP3 via its activity on PELP1 allows Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD + were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD + hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD + hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD + hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

reproduction in any medium, provided the original author and source are credited.

Data Availability Statement:

All relevant data are within the manuscript and its Supporting Information files.

Introduction

Several bacterial pathogens can circumvent host innate immune responses during infection, often by injecting effector proteins into host cells that target components of innate immune pathways. In many cases, these effectors contain eukaryotic-like domains capable of modulating receptor proximal events. This is the case of Toll/interleukin 1 receptor (TIR) domains present on the cytosolic faces of all Toll-like receptors (TLRs) and corresponding adaptor proteins, enabling the formation of a scaffold for the assembly of intricate protein signaling complexes [1]. The formation of these supramolecular organizing complexes (SMOCs) involves both self-interactions and interactions with other TIR domains [2]. TIR domains are also present in plants, where they mediate disease resistance, in amoebas with a role in ingestion of bacteria and immune-like functions, as well as in many bacterial genera [3]. Several Gram-negative and Gram-positive bacterial pathogens are known to rely on TIR domain-containing protein effectors for down-regulation of TLR-signaling during infection [4]. One of the best characterized is the TIR-containing protein of uropathogenic E. coli (TcpC), prevalent in clinical isolates associated with acute pyelonephritis in children. TcpC was shown to contribute to kidney pathology by hijacking the MyD88 TLR adaptor, resulting in inhibition of TLR4 and TLR2 signaling [5]. TcpC inhibition of TRIF-and IL-6/IL-1-dependent pathways has also been described [6]. Interestingly, the observation that expression of TirS from Staphylococcus aureus, present in a multi-drug resistant (MDR) island of numerous clinical isolates, is induced by specific antibiotic treatment [7] raises the possibility that these bacterial proteins may be tightly regulated, enhancing virulence, persistence or dissemination in particular clinical contexts such as exposure to selective pressure.

For some pathogens, additional functions have been assigned to bacterial TIR domains other than the downregulation of TLR pathways, as in the case of PumA from Pseudomonas aeruginosa, which interferes with TNF receptor signaling by targeting UBAP1 [8], a component of the endosomal-sorting complex required for transport I (ESCRT-I). Also, E. coli TcpC directly interacts with the NACHT leucine-rich repeat PYD protein 3 (NLRP3) inflammasome and caspase-1 resulting in inflammasome perturbation [9].

Author summary

Brucella is a genus of zoonotic bacteria that cause severe disease in a variety of mammals, ranging from farm animals (as bovines, swine and ovine) to marine mammals. Transmission to humans, often by ingestion of non-treated dairy products, leads to serious systemic infection. Brucella abortus invades host cells and replicates intracellularly. Such behavior relies on the injection of bacterial proteins into the host cytoplasm via specialized secretion systems. Our work focuses on the study of two of these factors, BtpA and BtpB, previously described to contain Toll/Interleukin-1 Receptor (TIR)-domains that modulate innate immunity. We use here two biological models: the yeast Saccharomyces cerevisiae and human cell lines. We found that the TIR domains of both Brucella proteins were necessary and sufficient to collapse energy metabolism in yeast by depleting ATP and NAD + . This result was translatable to higher cells and consistent with the recently described NADase activity of some TIR domains both in mammalian and bacterial proteins. Importantly, we demonstrate that Brucella down-regulates total NAD levels in host cells by using both BtpA and BtpB effectors. Our results show that NAD + is targeted by Brucella during infection, which may constitute a novel mechanism for its pathogenicity.

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Brucella TIR proteins deplete host cell NAD + Recent work on mammalian TLR adaptor SARM1 and plant nucleotide-binding leucinerich repeat (NLR) immune receptors, such as RUN1, unveiled that their TIR domains possess enzymatic activity [10,11]. Authors went on to demonstrate that not only eukaryotic but also prokaryotic TIR domains, in general, constitute a new family of nicotinamide adenine dinucleotide (NAD + ) hydrolase enzymes [12]. Although this NADase activity is efficiently neutralized in the bacteria by an unknown mechanism, when heterologously expressed in laboratory E. coli strains or assayed in vitro these prokaryotic TIR domains were able to cleave NAD + . Loss of NAD + was also detected when full-length S. aureus TirS was ectopically expressed in mammalian cultured cells [12].

One of the bacterial TIR domains shown to have NAD + -consuming activity when expressed in E. coli was that of BtpA (also known as TcpB) from Brucella spp. [12]. In Brucella abortus, a clear role in virulence has been established not only for BtpA but also for BtpB, the second TIR domain-containing protein of Brucella. Together, these effectors have been shown to downmodulate dendritic cell activation contributing to the stealthy characteristics of this pathogen in the context of chronic brucellosis [13,14].

The precise target of Brucella TIR-containing effector proteins remains unclear. BtpA has been proposed to act as a mimic of the TLR adaptor TIRAP by binding specific phosphoinositides of the plasma membrane [15] and increasing TIRAP ubiquitination and degradation during infection [16]. However, preferential binding to MyD88 was also demonstrated [17]. It is likely that these Brucella TIR-containing proteins display additional targets or functions, as they modulate microtubule dynamics when ectopically expressed [18,19] and BtpA was shown to induce the unfolded protein response [20].

Given all the possible roles proposed for these Brucella TIR effectors and their potential NADase activity we set out to investigate in greater detail their functions. By combining ectopic expression in the model eukaryotic organism Saccharomyces cerevisiae and human cells, as well as in vitro infection studies we have found that BtpA and BtpB reduce total NAD levels during infection, suggesting their NADase activities are an integral part of their role in Brucella pathogenesis. Our results point towards a novel function of these effectors in modulation of host metabolism through the modulation of intracellular NAD levels during infection.

Results

Expression of Brucella abortus TIR-domain containing BtpA and BtpB proteins in S. cerevisiae induces toxicity

To gain insight into the roles of BtpA and BtpB in modulation of cellular functions, btpA and btpB genes were cloned in a yeast expression vector under the control of the inducible GAL1 promoter to produce the corresponding GFP fusion proteins. Thus, expression was repressed in glucose-based media, but incubation of yeast transformants in galactose-based media led to the expression of GFP-BtpA and GFP-BtpB, as verified by Western blotting (S1A and S1B Both GFP-BtpA-TIR and GFP-BtpB-TIR conspicuous filaments resembled cytoplasmic microtubule bundles. However, immunofluorescence with anti-tubulin antibodies revealed that they did not co-localize with tubulin (S2 Fig) .  Although we cannot rule out that Brucella TIR domains are interacting with filamentous structures other than tubulin in yeast, our results suggest that they are prone to forming highly ordered structures by self-interaction, and that their N-terminal extensions negatively influence this behaviour.

BtpB depolarizes actin patches, blocks endocytosis and down-regulates signaling in S. cerevisiae

To understand the mechanisms underlying growth inhibition in yeast expressing Brucella TIR proteins, we chose to analyze the effects of full-length BtpB. The actin cytoskeleton supports polarized growth in yeast during budding, so growth arrest could be caused by actin dysfunction. Indeed, as shown in Fig 2A, staining of actin cortical patches with rhodamine-conjugated phalloidin revealed a dramatic loss of polarization of actin structures towards the growing bud and septum region. Moreover, the BtpB TIR domain was fully responsible for this phenotype (Fig 2A). Besides supporting growth along the budding cycle, actin function is important for endocytosis. We used the FM4-64 fluorochrome to monitor endocytic traffic. Internalization of this non-permeable molecule via the endocytic pathway leads to staining of the vacuolar membrane in cells after 1 hour of incubation [21]. We observed that cells that efficiently expressed GFP-BtpB, as judged by the presence of intense green fluorescent cytoplasmic spots, were unable to internalize this marker, as compared to those lacking green fluorescence or control cells expressing GFP alone (Fig 2B), indicating that BtpB severely blocked endocytosis. This phenotype also relies on BtpB TIR domain, as shown in S1C Fig.

Often, cellular stresses that lead to actin depolarization in yeast trigger the activation of signaling cascades involving mitogen-activated protein kinase (MAPK) modules, such as the cell wall integrity (CWI) pathway, engaging the Slt2 MAPK [22]. Also, we have previously described that some bacterial effectors, such as Salmonella SteC and SopB [23,24], depolarize actin by down-regulating small GTPases when expressed in yeast, leading to concomitant dephosphorylation of downstream Fus3 and Kss1 MAPKs of the mating pathway. Thus, we tested MAPK activation levels in BtpB-expressing cells by immunoblot using anti-phospho-MAPK antibodies. Peculiarly, all Slt2, Fus3 and Kss1 MAPK basal phosphorylation levels were downregulated in BtpB-expressing cells, but not upon BtpA expression (S3A Fig) . Then we investigated whether BtpB would be able to downregulate MAPK activation upon stimulation of these pathways, by incubation at 39 ºC or in the presence of the cell wall-stressing compound Congo red to stimulate the CWI pathway, and by using the mating pheromone α-factor to activate Fus3 and Kss1. Although BtpB still allowed activation of these pathways by the stimuli, MAPK phosphorylation was always less efficient (S3B Fig) . A fourth MAPK, Hog1, a p38 homolog, operates in budding yeast responding to high osmolarity challenges [25]. As observed for the other MAPKs, phosphorylation of Hog1 was less efficient in the presence of BtpB when this pathway was stimulated by osmotic stress (S3C Fig) . Since these MAPK pathways do not share upstream components, it is striking that they all were simultaneously downregulated by BtpB expression. Such a general effect in MAPK phosphorylation might reflect inability of the cell to properly undergo phosphorylation events. In support of this view, when heterologous mammalian Akt1 (which undergoes phosphorylation in its activation site by conserved yeast PDK-like kinases [26]), was co-expressed with BtpB, a reduced phosphorylation was also observed (S3D In sum, BtpB and BtpB-TIR expression in yeast result in severe actin disorganization, endocytic block and a general defect in the phosphorylation of all signaling kinases tested.

Genetic screen for yeast genes that suppress BtpB-induced lethality

We pooled three non-overlapping libraries obtained from the whole genome yeast ORF collection, consisting of all S. cerevisiae predicted ORFs cloned in an expression vector under the

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Brucella TIR proteins deplete host cell NAD + control of the inducible GAL1 promoter transformed in E. coli. This pooled whole genome expression library was co-transformed with a GFP-BtpB GAL1-based expression plasmid and positive selection allowed the recovery of genes suppressing BtpB toxicity in galactose-based medium. Suppressor genes listed in S4A Fig and S3 Table were selected when growth rescue (i) was confirmed after individual re-transformation, (ii) was specific for BtpB-induced growth inhibition, but not that of other toxic heterologous protein (PI3Kα-CAAX) [26], and (iii) was not due to a lower production of GFP-BtpB, as verified by immunoblot (S4B Fig) . As shown in S4A Fig, suppression was partial in all cases. Co-transformation of these suppressors with BtpB-TIR led to the same rescue levels, although no growth recovery was detected when coexpressed with BtpA-TIR (S4C and S4D Fig) . Thus, either these suppressors are specific for BtpB-TIR domain derived toxicity in yeast or the effect of BtpA-TIR is too strong to allow partial suppression. Although most of these genes have not been yet assigned a bona fide function in yeast, a subset of them, INM2, RBK1, and DOG2 are sugar or inositol phosphorylating/ dephosphorylating enzymes related to metabolic pathways (S3 Table ). DOG2 encodes a 2-deoxyglucose-6 phosphate phosphatase and its overexpression overcomes toxicity of this glycolytic inhibitor [27], and RBK1 encodes a putative ribokinase, which has been recently shown to be catalytically active [28]. These results suggest that metabolic shifts related to carbon source usage partially counteract BtpB toxicity.

BtpA and BtpB deplete ATP and NAD + in the yeast cell

Our results on BtpB expression in yeast affecting dynamic cellular events such as cytoskeletal function and vesicle traffic as well as general kinase function would be consistent with limiting intracellular ATP levels. Furthermore, the fact that sugar kinase/phosphatases were isolated as btpB overexpression suppressors suggest that energetic metabolism is compromised in BtpBexpressing yeast cells. Recently, Essuman et al. [12] reported that the TIR-domain of proteins from phylogenetically diverse bacteria, including Brucella BtpA, displayed enzymatic activity as NAD + hydrolases. Thus, we were prompted to study ATP and NAD + levels in yeast cells expressing BtpA and BtpB. Yeast cells expressing BtpB or the TIR domains of either BtpB or BtpA, showed significant losses of both ATP and NAD + intracellular levels, as determined by luciferase assay or quantitative mass spectrometry respectively (Fig 3). This effect was especially dramatic in NAD + levels, which were lowered about one order of magnitude upon BtpB overexpression. We also observed a slight but significant reduction of NAD + in the case of fulllength BtpA (Fig 3B). The decrease in NAD + and ATP correlated very well with the differential toxicity for yeast cells of each protein version (Fig 1A), as full-length BtpB had the strongest effect on intracellular NAD + and ATP levels while, in the case of BtpA, the TIR domain alone had a more dramatic effect than the full-length protein. This raises the idea that the N-terminal extension of BtpA, but not that of BtpB, has a negative regulatory effect on the C-terminal TIR/NAD + hydrolase domain.

As a control, we generated a BtpB E234A catalytically inactive mutant, by changing to Ala the equivalent Glu residue described by Essuman et al. [12] to be essential for catalysis in other TIR domains. BtpB E234A was no longer toxic for yeast (Fig 4B) In agreement with its lower toxicity, this BtpB mutant was expressed at higher levels than the toxic wild-type version (S1B Fig) . Expression of the BtpB E234A mutant had no effect on ATP or NAD + intracellular levels (Fig 3), strongly suggesting that, as described for other TIR domains [12], this residue is essential for the catalytic activity of BtpB.

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Brucella TIR proteins deplete host cell NAD +

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Brucella TIR proteins deplete host cell NAD + 0.0046 ( ) for BtpB-TIR. (B) Cellular NAD + levels measured by mass spectrometry, standardized as a NAD + /extract protein ratio, from YPH499 cells transformed with the same plasmids as in A. Graph shows NAD + /protein ratio as a percentage of the empty vector control cells NAD + /protein ratio. Data correspond to means ± standard deviation of four different transformants and statistical comparison was done with one-way ANOVA with p-values referred to vector <0.0001 ( ) for BtpA-TIR, BtpB and BtpB-TIR, and 0.0134 ( ) for BtpA. https://doi.org/10.1371/journal.ppat.1007979.g003

Mapping of residues essential for NAD + hydrolase function at the TIR domain of BtpB

Taking advantage of the severe toxicity of BtpB in yeast, we devised a screen for the isolation of loss-of-function mutations by random mutagenesis. This was performed by plasmid gaprepair [29], forcing in vivo homologous recombination between an open gapped plasmid and a partially overlapping insert encoding BtpB, which had been generated by error-prone PCR. Such strategy allows direct selection in galactose-based medium for recombinant clones bearing mutations in btpB that yield the protein non-toxic. Ten single and two double mutants were recovered and sequenced (S4 Table ). As shown in S5A Fig, most mutations corresponded to non-conservative amino acid changes in highly conserved regions between BtpA and BtpB TIR domains. Some of these residues are also conserved in the TIR-domain of human SARM1 and plant RUN1, in which the NAD + hydrolytic activity was recently described [10,11].

To decipher the effects of the mutations on BtpB properties, we mapped the corresponding residues on the BtpA-TIR domain structure (PDB: 4LZP) [30], as BtpB structure is not yet solved. As seen in Fig 4A, none of the residues mutated belonged to the TIR-TIR interface. S162 (S149 in BtpA) and F163 (H150 in BtpA) belong to the pA strand. Y225 (F208 in BtpA) and Q226 (F209 in BtpA) are part of the small helix αC. Mutations of these residues are likely to disrupt the inner core and thus destabilize the whole structure.

Mutation of BtpB S201P (S185 in BtpA) is likely to perturb the NAD+ catalytic site. In the recent crystal structure of NADP + -bound RUN1-Tir domain [11] (PDB: 6O0W), the substrate lies in a pocket formed by the BB-loop and the loop containing the conserved catalytic WxxxE motif [19] (S5B Fig) . In BtpA structure S185 lies in the BB loop and interacts with the W213 (W231 in BtpB) of the WxxxE motif, which contains the essential catalytic E217 residue (E234 in BtpB). Finally, D158 (D145 in BtpA), F188 (F174 in BtpA), Y193 (Y178 in BtpA), and I291 (I275 in BtpA) residues clustered in two patches at the protein surface (Fig 4A).

In order to determine whether NAD+ hydrolase and filament formation of TIR domains were separable features, we transferred mutations D158G, S162P and Y255C, as well as the mutation in the catalytic residue E234A, to GFP-BtpB-TIR to study whether loss of toxicity corelated with the ability of the TIR domain alone to produce filaments. Interestingly, only E234A, S162P and, partially, Y225C mutations eliminated BtpB-TIR toxicity in yeast cells (Fig 4B), despite the fact that all four mutations fully prevented toxicity and endocytosis defects in full-length BtpB (S5C-S5E Fig) . Moreover, only the GFP-BtpB-TIR S162P mutant significantly lost the ability to form protein filaments (Fig 4C and4D), probably because that mutation damaged the inner core (Fig 4A) These results indicate segregation between filament formation and growth inhibitory functions of BtpB-TIR in yeast and highlight the importance of the Glu234 residue specifically for NAD + hydrolase activity, while Ser162 is key for both features.

We also changed by site-directed mutagenesis the catalytic E217 residue to Ala in both fulllength and the TIR domain alone of BtpA. Both mutants lost their toxicity on yeast (Fig 4E). Moreover, BtpA-TIR E217A did not reduce MAPK phosphorylation and yeast cells sustained higher levels of expression as compared to WT BtpA-TIR (S1A Fig) . Although a statistically significant reduction in the percentage of cells showing BtpA-TIR filaments was found (45.1%

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Brucella TIR proteins deplete host cell NAD +

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Brucella TIR proteins deplete host cell NAD + from pYES2 plasmid derivatives, under control (Glucose) and induction (Galactose) conditions. (C) Normarski and fluorescence microscopy of YPH499 cells expressing GFP-BtpB-TIR and the indicated mutants, after 4h induction. Scale bars correspond to 5 µm. (D) Graph displaying percentage of cells showing filamentous fluorescent structures. Data corresponds to means ± standard deviation of three independent transformants and statistical comparison was done with one-way ANOVA with a p-value < 0.0001 ( ) between BtpB-TIR WT and S162P. (E) Ten-fold serial dilution growth assay of YPH499 yeast strain bearing pYES2 empty vector and pYES2 plasmid derivatives expressing BtpA, BtpA-TIR and their corresponding catalytically inactive mutants E217A, under control (Glucose) and induction (Galactose) conditions. (F) Normarski and fluorescence microscopy of yeast cells expressing pYES2-GFP-BtpA-TIR and its E217A mutant version. Scale bars correspond to 5 µm. https://doi.org/10.1371/journal.ppat.1007979.g004 ± 7.7 for BtpA-TIR vs. 26% ± 12 for BtpA-TIR E217A), these structures were larger and more intense for the mutant than for the wild type version (Fig 4F). Importantly, these results indicate that, as observed for BtpB, the catalytic E217 residue is essential for toxicity in yeast but still allows assembly of the BtpA TIR domain into ordered structures.

Inhibition of endocytosis occurs upon ectopic expression of BtpB in human cells but not during infection

To investigate whether the results obtained in yeast were translatable to mammalian cells, we began by overexpressing BtpB in human epithelial cells (HeLa). As previously described [19] and consistent with the yeast model, a punctate accumulation of BtpB was observed in the cytosol of HeLa cells. These results were obtained for GFP-BtpB (Fig 5), as well as Mycexpressing BtpB (S6A Fig) , indicating that this localization is independent of the tag. To gain insight into the type of structures BtpB was forming, we labelled for different endocytic markers. Some of these structures were enriched in mono-and poly-ubiquitinated proteins as recognized by the FK2 antibody (Fig 5A), which could correspond to either aggregates of misfolded protein or sites with densely ubiquitinated proteins as previously described [19]. However, some of the BtpB compartments did not show labelling with the FK2 antibody and were also negative for the lysosomal associated membrane protein Staphyloccocus aureus TirS protein [7]. Most likely, as also inferred from the above yeast data, these structures correspond to self-assembled ordered filaments consisting of the TIR domain, which are absent when stabilized by the presence of the N-terminal domain. These results confirm that the N-terminal portion of BtpB plays an important role in subcellular localization. Interestingly, the BtpB E234A mutant retained the dot-like distribution observed for BtpB in HeLa cells (S6E Fig).

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Brucella TIR proteins deplete host cell NAD + We next determined if BtpB expression resulted in perturbation of endocytosis in human cells as observed in yeast. Fluorescently labeled transferrin or Epidermal Growth Factor (EGF) were incubated with HeLa cells expressing GFP-BtpB or GFP alone and the percentage of cells with uptake of these endocytosis markers quantified by microscopy. The expression of GFP-BtpB significantly decreased endocytosis of both markers in comparison to GFP alone (Fig 7A -7C) consistent with the yeast model.

Although overexpression of individual effectors provides a powerful tool to investigate direct functions of these proteins, to thoroughly investigate the capacity of BtpA and BtpB to inhibit endocytosis, we analyzed this phenotype during infection. Although we could observe a slight decrease of endocytosis after 24 h of infection of HeLa cells, this phenotype was neither statistically significant nor abrogated by deletion of btpA nor btpB (S7A Fig) . Furthermore, no significant differences were observed at 48 h post-infection for HeLa cells infected with WT Brucella in comparison to cells infected with mutants lacking either btpA, btpB or both genes (Fig 7D). Finally, no impact on endocytosis was observed in immortalized bone marrow-derived macrophages (iBMDM) at 24 nor 48 h post-infection (S7B Fig and Fig 7E,respectively), suggesting that Brucella TIR proteins do not interfere with endocytosis during infection.

BtpA and BtpB deplete cellular NAD when ectopically expressed in human cells and during infection

As previous studies attributed a NAD + -consuming activity to the BtpA TIR domain when expressed in E. coli [12] and our experiments using the eukaryotic yeast model showed that both BtpB and BtpB-TIR strongly reduce intracellular NAD + content, we next assessed total NAD levels in HeLa cells expressing either Myc-BtpB or Myc-BtpA in comparison to Myc alone by using a colorimetric assay. Both Myc-tagged proteins were well expressed in epithelial cells although BtpA always migrated as a double band, potentially indicative of post-translational modifications occurring in the cell ( To determine whether Brucella could impact intracellular NAD levels during infection, we first established that all bacterial strains had equivalent levels of total NAD in the inocula (S7C Fig), which corresponds to a 16h culture, the time required to reach early stationary phase used for our infection studies. We next infected HeLa cells with wild-type or mutant strains lacking either btpA or btpB and quantified the levels of total NAD. Although we did not observe any differences at 24h post-infection, we could observe that B. abortus infection for 48h resulted in reduction of total NAD levels in a manner dependent on BtpA and BtpB (Fig 9A). As it is well established that btp mutants replicate to the same levels as wild-type Brucella [14] and we have found they have equivalent bacterial total NAD levels (S7C Fig) we can conclude that BtpA and BtpB NAD-consuming activities are likely to impact host intracellular NAD levels. To confirm these phenotypes were specifically due to the absence of BtpA and BtpB, we attempted to complement the mutant strains. The phenotype of the btpA mutant strain could be restored by expressing btpA from a plasmid (Fig 9B). Although the same tendency could be observed for the complementation of the btpB mutant, due to a lower effect on the NAD concentration in HeLa cells infected with the btpB mutant we could not obtain statistical significance with the number of experiments performed (Fig 9C). We therefore infected iBMDM, as a much higher rate of infection can be attained with phagocytic cells. In this cellular model, wild-type B. abortus infection also resulted in the reduction of intracellular NAD levels, in a manner dependent on BtpA and BtpB. The expression of each gene from a plasmid

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Brucella TIR proteins deplete host cell NAD +

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Brucella TIR proteins deplete host cell NAD + 48 h with either wild-type B. abortus or a mutant strain lacking btpA, btpB or both genes. Cells were then incubated with EGF conjugated with Alexa Fluor 555 for 10 minutes and the percentage of infected cells showing uptake of this fluorescent marker quantified by microscopy. Counts correspond to individual microscopy fields, with a total of at least 200 cells counted for each, from three independent experiments. Mock infected cells are included as a control. Data correspond to means ± standard deviation and statistical comparison was done with one-way ANOVA test, with no statistical significance observed. Finally, to determine if the observed reduction of NAD was due to the catalytic activity of the TIR domain we complemented the btpA mutant with a plasmid carrying a E217A mutation in btpA (∆btpApbtpA E217A) and the btpB mutant with a plasmid expressing a E234A mutation in btpB (∆btpBpbtpB E234A). We first controlled that these catalytic mutant versions of BtpA and BtpB could be efficiently translocated into host cells. We constructed TEM1 fusions as previously reported [14] and determined the percentage of cells emitting coumarin fluorescence at 24 h post-infection (S7D Fig) . We had to use RAW macrophages for these experiments as previously described [14] because CCF2 was toxic for iBMDM. In the case of BtpA we observed a level of translocation of TEM-BtpA E217A consistent with what was previously reported for the wild-type TEM-BtpA [14]. In the case of BtpB, a lower percentage of infected cells showed translocation of the TEM-BtpB E234A, consistent with what has been observed for the wild-type TEM-BtpB (less than 2% of infected cells) [14].

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Brucella TIR proteins deplete host cell NAD + ΔbtpApbtpA 0.0124, ∆btpApbtpA versus ΔbtpApbtpA E217A 0.0177 and wild-type versus ΔbtpApbtpA E217A 0.0072. (C) HeLa cells were infected for 48 h with either wild-type B. abortus, a ∆btpB mutant, the complemented strain ∆btpBpbtpB and a ∆btpB complemented with a catalytic mutant ∆btpBpbtpB E234A. Results are normalized to mock infected cell values and correspond to means ± standard deviation from three independent experiments and statistical comparison was done with a one-way ANOVA test, with a slight statistical significance only observed between wild-type and ∆btpB (p = 0.0491).

https://doi.org/10.1371/journal.ppat.1007979.g009

We next measured the concentration of total NAD in both HeLa and iBMDM infected cells. In HeLa cells, catalytically inactive BtpA failed to complement the mutant strain (Fig 9B). Consistently, this is also the case in iBMDM for both BtpA and BtpB (Fig 10A and10B). Together these results indicate that BtpA and BtpB contribute to depletion of intracellular total NAD levels via direct enzymatic cleavage of this metabolic co-factor during infection, assigning a novel function for these two effectors during Brucella infection.

Discussion

Bacterial TIR domain-containing proteins have been shown to be major contributors to the evasion of innate immunity for a variety of bacterial pathogens, mainly by interfering with the assembly of innate immune signaling complexes involving TIR domains [4]. However, certain

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Brucella TIR proteins deplete host cell NAD + TIR domains have recently been demonstrated to possess a NAD + hydrolase activity which may contribute to their function, as for example the case of mammalian SARM1 [12] or plant NLR immune receptors [11]. We have found that both the Brucella TIR domain-containing proteins BtpA and BtpB retain this NAD + hydrolase activity inside cells when ectopically expressed in yeast or human cells, as well as during infection, resulting in reduction of intracellular total NAD levels at late stages of the infection. Furthermore, we have highlighted that the N-terminal non-TIR domains of these proteins are necessary for intracellular targeting of the effectors. Remarkably, BtpA-TIR and BtpB-TIR resulted in formation of long filament-like structures when ectopically expressed in yeast and human cells. Since this phenomenon can only be observed in the absence of their N-terminal regions it is likely that such N-terminal extensions may function to modulate intrinsic TIR self-assembly. Furthermore, genetic analyses in yeast revealed that such highly ordered structures formed by expression of the TIR domains alone are still achieved when expressing point mutants that lose their NADase activity, suggesting that distinct features of TIR domains rely in different structural determinants.

NAD + is an important coenzyme participating in hundreds of enzymatic reactions, notably glycolysis, the TCA cycle and mitochondrial oxidative phosphorylation. NAD + homeostasis is essential for metabolic balance and cell survival either being used as an electron carrier in redox reactions or being consumed as a substrate for numerous reactions. Beyond its wellknown role in bioenergetics, NAD + has been found to have a prominent function in cell signaling, with sirtuins, poly ADP-ribose polymerases (PARPs) and CD38 using NAD + as substrate [31,32]. NAD + has also been shown to be a key modulator of immune metabolism, acting as an important metabolic switch. In macrophages, it has been shown that increased NAD + levels are associated with activation and control of inflammatory responses, particularly involving regulation of TNFα transcription in classically activated pro-inflammatory (M1) macrophages [33,34]. Interestingly, NAD + limitation also prompts important cellular changes such as the Warburg effect, a cellular state in which consumption of glucose is increased and aerobic glycolysis is favoured instead of the more energy efficient mitochondrial oxidative phosphorylation [35]. A switch to Warburg metabolism has also been observed upon immune activation of many cell types, for example macrophages, following pattern recognition receptor activation [36]. In addition, low NAD + levels are a trigger for cell death via necroptosis in macrophages [37].

Our work highlights that Brucella is decreasing total NAD levels in the host cell, likely contributing to modulation of cellular metabolism and signaling. This is dependent on two translocated effectors, BtpA and BtpB, containing a TIR domain that had previously been shown to down-modulate innate immune signaling in specific in vitro differentiated mouse bone marrow-derived dendritic cells [13,14]. It is possible that the two phenotypes, NAD reduction and blocking of TIR-TIR interactions along the TLR signaling pathways are intimately connected. Indeed, targeting of these Brucella effectors to the vacuolar membrane or innate immune signaling platforms might locally impact NAD + levels inhibiting specific enzymatic reactions. Interestingly, the first enzyme to use NAD + in glycolysis, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been shown to be recruited to the membrane of Brucella-containing vacuoles playing an essential role in intracellular replication [38]. Previous studies have also reported the role of the specific T4SS effector BPE123 in targeting the host enolase, another enzyme of the glycolysis pathway, which was also shown to be essential for Brucella intracellular multiplication in human cultured epithelial cells [39].

Host metabolism during Brucella infection has only recently started to be unravelled. In classically activated macrophages, Brucella infection was shown to induce a Warburg-like effect, with high consumption of glucose and generation of lactate efficiently used as a carbon source by intracellular replicating bacteria [40]. Interestingly, in alternatively activated

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Brucella TIR proteins deplete host cell NAD + macrophages, abundant during chronic brucellosis, a shift from oxidative metabolism of glucose to oxidation of fatty acids occurs, enhancing the availability of glucose to promote intracellular bacterial replication [41]. In this study, we now highlight the role of the innate immune regulator effectors BtpA and BtpB in direct control of host energy metabolism.

Both BtpA and BtpB TIR domains were robust enough as NADases in the yeast heterologous model as to drop NAD + levels over one order of magnitude, causing a severe decrease of ATP availability in the cell and strong toxicity when overexpressed. The fact that full length BtpA is not as toxic for the yeast cell suggests that the N-terminal domains of BtpA negatively regulate BtpA catalytic activity. BtpB, on the contrary, is intrinsically active both in the absence and in the presence of its N-terminal extension. We show here that catalytically dead mutants in BtpA and BtpB TIR domains, still have the ability to self-aggregate and form filaments, proving that both features can be segregated. BtpA has been related to tubulin structures in host cells, specifically by protecting microtubules from depolymerization [19,42]. However, we did not see coincidence of TIR cytoplasmic filaments with tubulin. ATP is necessary to achieve depolymerization of microtubules by nocodazole [43], so NAD + depletion and low ATP levels could contribute to the microtubule-stabilizing properties assigned to BtpA. Similar ATP-dependent phenomena could account for the inhibition of endocytosis in yeast and human cells. It is important to note however, that no impact of BtpA and BtpB on endocytosis was observed in infected cells. Therefore, ectopic over-expression of these effectors and its strong effect on ATP and NAD could be responsible for this phenotype, absent when a much smaller amount of protein is translocated into host cells during infection. Alternatively, Brucella infection may induce compensatory effects that would mask this phenotype, for example translocate other effectors that would enhance endocytosis.

Why Brucella may limit NAD + levels and energy metabolism in particular subcellular compartments and stages of the establishment of the intracellular niche? This is a challenging question and at this stage we can only speculate. NADase activity may contribute to evading the innate immune response. Importantly, NAD + levels are sensed by sirtuin proteins, like SIRT1, leading to the activation of several signaling pathways, some related to immunomodulation [44]. NAD + -dependent SIRT1 has recently been shown to be an important hub for cellular defence against M. tuberculosis intracellular survival. M. tuberculosis infection reduces intracellular NAD + and down-regulates SIRT1, which can be reversed by the addition of SIRT1-activating compounds that represent a potential therapeutic option [45]. The Tuberculosis Necrotizing Toxin (TNT) of M. tuberculosis bears NAD + glycohydrolase activity, which was shown to be important for mycobacterial replication in macrophages, and is involved in triggering necroptosis as a consequence of NAD + depletion [37,46]. In addition, it has been proposed that reduced NAD + and ATP levels in Salmonella-infected macrophages, a process dependent on the Salmonella Pathogenicity Island 2 (SPI-2) type 3 secretion system [47], trigger the modulation of TORC1 to protect intracellular bacteria from xenophagy via lysosomal degradation of its upstream SIRT1/LKB1/AMPK regulators [47]. Thus, NAD + depletion could eventually be understood as a strategy to evade cellular innate immunity responses and promote bacterial intracellular survival.

To our knowledge, this is the first report on bacterial TIR domain-containing proteins showing NAD + hydrolase activity on the host cell during infection. Our results on the comparison of the divergent behaviour of full length and TIR domains alone in the case of BtpA and BtpB, together with the observation that the N-terminal non-TIR extensions determine subcellular localization and prevent filament formation, suggests that these extensions may finely tune NADase activity in the context of infection, likely by negatively modulating TIR-TIR assembly and by directing NADase activity to specific intracellular compartments. Further work is now necessary to better understand the control of NAD + homeostasis during Brucella

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Brucella TIR proteins deplete host cell NAD + infection. Knowledge on how metabolic switches occur during infection at a molecular level could provide clues for the development of therapeutic strategies and vaccines.

Materials and methods

Saccharomyces cerevisiae strains and growth conditions

YPH499 (MATa ade 2-101 trp1-63 leu2-1 ura3-52 his3-200 lys2-801) [48] was the S. cerevisiae strain for general use in these studies, unless otherwise stated. W303-1A (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) [49] was used for the Yeast ORF overexpression library screening. The E. coli strain DH5α F 0 (K12∆(lacZYA-argF)U169 deoR supE44 thi-1 recA1 endA1 hsdR17 gyrA96 relA1 ( §80lacZΔM15)F 0 ) was used for general molecular biology. As a general medium, YPD (1% yeast extract, 2% peptone and 2% glucose) broth or agar was used for growing yeast cells. For plasmid selection, synthetic complete medium (SC) contained 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulphate, 2% glucose and the appropriate amino acids and nucleic acid bases supplements. SG and SR were SC with 2% galactose or 1.5% raffinose, respectively, instead of glucose. For galactose induction experiments in liquid media cells were grown in SR medium to log phase and then galactose was added to 2% for 4-6 h. Effects of the expression of Brucella genes on yeast growth were tested by ten-fold serial dilution assay: spotting cells onto SC or SG plates lacking the appropriate auxotrophic markers to maintain the corresponding plasmids, and incubating them at 30 ºC for 72 h [26].

Cell culture and transfections

HeLa cells (from ATCC) were grown in DMEM supplemented with 10% of fetal calf serum (FCS) and were transiently transfected using Fugene (Roche) for 24 h, according to manufacturer's instructions. Immortalized bone marrow-derived macrophages from C57BL/6J mice were grown in DMEM supplemented with 10% of fetal calf serum (FCS) and 10% of spent supernatant of L929 cells (that provides M-CSF). RAW 264.7 macrophages (ATCC) were grown in DMEM supplemented with 10% of fetal calf serum (FCS). The Brucella abortus 2308 strain was used for transfection and genetic manipulation.

Construction of yeast expression plasmids

General molecular biology techniques, as well as transformation of yeast by the lithium acetate method were performed according to standard procedures. All plasmids and oligonucleotides used in this work are listed in S1 and S2 Tables respectively. pYES2-GFP, pYES3-GFP-Akt1 and YCpLG-PI3Kα-CAAX were previously described [26,29,50]. To clone B. abortus btpA into the URA3-based pYES2-GFP plasmid, this gene was amplified from pGEM-T-easy-BtpA plasmid [13]. The primers used for amplification of btpA (named BtpA-UP and BtpA-LO) had BamHI respectively and EcoRI sites. PCR products were cleaved by these restriction enzymes to be inserted in the same sites of pYES2-GFP, generating the pYES2-GFP-BtpA plasmid. To obtain the pYES2-GFP-BtpB construction, btpB was amplified form pGEM-T-Easy-BtpB plasmid [13]using primers BtpB-UP and BtpB-LO, cleaved with BamHI and XbaI restriction enzymes and the insert obtained was cloned into the pYES2-GFP plasmid. pYES3-GFP-BtpB was generated on a similar way but using primers BtpB-UP-pYES3 and BtpB-LO-pYES3 and BamHI sites to clone into the TRP1-based pYES3-GFP plasmid. The latter plasmid had been constructed by subcloning the GFP sequence of pYES2-GFP with HindIII/BamHI sites into pYES3 (Invitrogen).

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Brucella TIR proteins deplete host cell NAD +

In order to obtain the truncated versions of both BtpA and BtpB, we generated pYES2-GFP-BtpA-N (1-126), pYES2-GFP-BtpA-TIR (127-275), pYES2-GFP-BtpB-N (1-139) and pYES2-GFP-BtpB-TIR (140-292) by amplifying the corresponding DNA fragments from pYES2-GFP-BtpA and pYES2-GFP-BtpB using for the N-terminal regions BtpA-UP + BtpA-126stop-LO and BtpB-UP + BtpB-140stop-LO primers, respectively. In the case of the C-terminal regions, BtpA-BamHI127-UP + BtpA-LO and BtpB-BamHI140-UP and BtpB-LO primers were used. All upper primers carried BamHI restriction site and lower primers had EcoRI site, except in the case of BtpB-LO, which carried an XbaI sequence. The PCR products were cleaved with their corresponding restriction enzymes and inserted in the same sites in the pYES2-GFP plasmid. Additionally, both C-terminal regions were also cloned into pYES3-GFP vector: BtpA-TIR (127-275) fragment was directly subcloned from pYES2-GFP-BtpA, whereas BtpB-TIR (140-292) was PCR amplified with BtpB-BamHI140-UP and BtpB-EcoRI-LO primers and then inserted on BamHI-EcoRI sites on pYES3-GFP.

DpnI-based site directed mutagenesis was performed using the QuikChange kit (Agilent). To generate the catalytically inactive mutants in both full length and TIR domain of BtpA and BtpB in the pYES2-GFP and pYES3-GFP backbones, the mutations were introduced with primers Mut-BtpAE217A-UP and Mut-BtpAE217A-LO and Mut-BtpBE234A-UP and Mut-BtpBE234A-LO respectively. To introduce the three selected loss-of-function BtpB mutations into the TIR domain construct, primers Mut-BtpBD158G-UP andMut-BtpBD158G-LO, Mut-BtpBS162P-UP and Mut-BtpBS162P-LO, or Mut-BtpBY225C-UP and Mut-BtpBY225C-LO were used.

Construction of mammalian expression vectors

The DNA fragment encoding amino acid residues 1-139 of BtpB (BtpB-N), 140-292 of BtpB (BtpB-TIR) and BtpB full-length from Brucella abortus were cloned into the Gateway pDONR (Life Technologies, ThermoFisher Scientific) and then cloned into the pENTRY (Life Technologies, ThermoFisher Scientific) GFP vectors according to the manufacturer. The following primers were used: BtpB Fw, BtpB Rv, BtpB-TIR Fw and BtpB-N Rv. BtpB E234A was constructed as above from the pYES3-GFP-BtpB E234A with primers used to amplify BtpB.

Construction of Brucella complementing vectors

The DNA fragments encoding btpA, and btpB were cloned into the plasmid pBBR-1-MCS4. The primers used for amplification of btpA had for the forward primer a SpeI restriction site and for the reverse primer a EcoRI restriction site. The primers used for amplification of btpB had for the forward primer a SacI restriction site and for the reverse primer a SpeI restriction site. The following primers were used BtpA Fw-pBBR-1-MCS4, BtpA Rv-pBBR-1-MCS4, BtpB Fw-pBBR-1-MCS4, BtpB Rv-pBBR-1-MCS4. The PCR products were cleaved with their corresponding restriction enzymes and inserted in the same site in the digested pBBR-1-MCS4 plasmid. The BtpA E217A and BtpB E234A complementation vectors were obtained from pBBR-1-MCS4-BtpA and pBBR-1-MCS4-BtpB, respectively using QuickChange Site-Directed Mutagenesis.

The mutations were introduced with primers BtpAE217A Fw, BtpAE217A Rv, BtpBE234A Fw, BtpBE234A Rv.

To clone BtpA E217A and BtpB E234A into the pFlag-TEM, this genes were amplified from pBBR-1-MCS4-BtpA E217A and pBBR-1-MCS4-BtpB E234A, respectively. The primers used for amplification of btpAE217A and btpBE234A had for the forward primer a XbaI restriction site and for the reverse primer a PstI restriction site. The PCR products were cleaved with their corresponding restriction enzymes and inserted in the same site in the pFlag-TEM plasmid.

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Brucella TIR proteins deplete host cell NAD +

The primers sued were: TEM-BtpAE217A FW-pFlagTEM, TEM-BtpAE217A Rv-pFlagTEM, TEM-BtpBE234A FW-pFlagTEM, TEM-BtpBE234A Rv-pFlagTEM.

Yeast cells microscopy and immunofluorescence

For fluorescence microscopy of live yeast to visualize GFP, cells were cultured in SR medium for 18 h at 30 ˚C, then, the appropriate amount of these cultures was suspended into fresh SG to reach an OD600 of 0.3, and they were incubated for additional 4-6 h for GAL1 promoter induction. Cells were harvested by centrifugation and observed directly. To monitor vacuolar morphology and endocytosis, staining with FM4-64 was performed as described [21]. Nuclear labelling was performed by adding DAPI at 1:1000 directly to the harvested cells in vivo and washed once with PBS. To observe actin, yeast cells were fixed and treated with rhodamineconjugated phalloidin (Sigma) as described [23].

Indirect immunofluorescence on yeast cells was performed as previously described [51]. Antibodies were used as follows: As primary antibody, monoclonal rat anti-alpha-tubulin (Serotec, YOL1/34) at 1:500 dilution; as secondary antibody, Alexa Fluor 594 anti-rat dye (Life Technologies) at 1:1000 dilution. DAPI was added at 1:1000 for nuclear labelling. Cells were examined in Eclipse TE2000U microscope (Nikon, Tokyo, Japan) and digital images were acquired with an Orca C4742-95-12ER charge-coupled-device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and processed by HCImage and ImageJ software.

HeLa cells immunofluorescence, antibodies and microscopy

Cells were fixed in Antigenfix (DiaPath), at room temperature for 10 min or methanol at -20 ˚C for 3 min for tubulin staining. Cells were then labelled at RT with primary antibody mix diluted in 0.1% saponin in PBS with 1% BSA and 10% horse serum for blocking. Primary antibody was incubated for 1 h followed by two washes in 0.1% saponin in PBS. Secondary antibodies where then mixed and incubated for a further 30 min, followed by two washes in 0.1% saponin in PBS, one wash in PBS and one wash in distilled water before mounting with Prolong Gold. Samples were examined on a Zeiss LSM800 laser scanning confocal microscope for image acquisition. Images of 1024×1024 pixels were then assembled using ImageJ.

Primary antibodies used were mouse anti-beta-tubulin clone E7 (Developmental Studies Hybridoma Bank) at 1:250 or mouse anti-vimentin (V9) at 1:100 (Sigma). Secondary antibodies used were anti-rabbit or mouse conjugated with Alexa Fluor 488, 555 or 647 all from Jackson Immunoresearch. When necessary phallodin-568 (1:1000) was used to label the actin cytoskeleton and DAPI nuclear dye (1:1000) for the host cell nucleus. When indicated, cytochalasin D was added for 2 h at a final concentration of 1 µg/ml.

HeLa cells endocytosis assay

Transferrin conjugated with Alexa Fluor 568 (Invitrogen) and EGF conjugated with Alexa Fluor 555 (Invitrogen) were added at 10 µg/ml and 50 µg/ml respectively, for 10 min a 37 ˚C. Cells were then placed on ice, washed with ice cold PBS twice and fixed in 3% paraformaldehyde for 15 min, followed by three washes with PBS.

Immunodetection by western blotting

Standard procedures were used for yeast cell growth, collection, breakage, protein separation by SDS-PAGE, and transfer to nitrocellulose membranes. Anti-P-MAPK antibody (Anti-phospho-p44/ p42 MAPK (Thr-202/Tyr-204), New England Biolabs) was used to detect dually phosphorylated Slt2, Kss1 and Fus3 MAPKs diluted 1:1000. Slt2 protein was detected using a

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Brucella TIR proteins deplete host cell NAD + polyclonal anti-Slt2 antibody [52], diluted 1:1000. To detect phosphorylated Hog1 high osmolarity pathway MAPK, antibody Anti-P-p38 (Sigma) at 1:1000 was used. Heterologous expressed Akt1 was detected with Anti-Akt1 (Cell Signalling) as total protein and with anti-P-Akt1(Thr)308 (Cell Signalling) for the phosphorylated forms. GFP fusion proteins were detected using monoclonal anti-GFP antibodies (Living Colors, JL-8) diluted 1:2000. As loading control either a monoclonal anti-actin (MP Biomedicals) diluted 1:2000 or a yeast specific polyclonal anti-Glucose-6-Phosphate Dehydrogenase (Sigma) diluted 1:50000 were used. In all cases, primary antibodies were detected using IRDye-680 or -800 anti-rabbit or anti-mouse antibodies (Li-Cor Biosciences), or Alexa-680 anti-mouse (Invitrogen) with an Odyssey Infrared Imaging System (Li-Cor Biosciences).

Yeast whole genome ORF overexpression library screening

A pooled S. cerevisiae whole genome ORF library (Yeast ORF collection, GE Healthcare), 4500 URA3-based plasmids, for overexpression under GAL1 promoter and protein A-tagged, was split into three groups. W303-1A wild type yeast strain (was co-transformed with pYES3-GFP-BtpB and one of the three library pools (S3A Fig) . BtpB toxicity suppression by overexpression of a specific cDNA was tested by its ability to grow in SG agar plates. 20 different candidates were selected and tested for specificity by co-transformation with YCpLG-PI3Kα-CAAX [26], another toxic construct for yeast cells that acts by a different mechanism. Eventually 7 positive ORF, listed on S3 Table, were found to specifically rescue BtpB toxicity.

Random mutagenesis of btpB and isolation of mutants

The region of pYES2-GFP-BtpB including amino acids 118 to 292 delimited by the mutazarUP and mutazarLO primers was amplified by PCR using low-fidelity Taq DNA Polymerase (Biotools) under standard conditions. The PCR products were purified with a QIAquick Gel Extraction kit (250) kit (Qiagen) and 5 µg of DNA were co-transformed into YPH499 yeast cells with 1 µg of the largest fragment of pYES2-GFP-BtpB plasmid, resulting from digestion with BsiWI/XbaI. Such BsiWI/XbaI digestion of pYES2-GFP-BtpB produces a gap, so the btpB allele can only be reconstructed upon recombination with the amplicon by in vivo gap repair. Recombinants were recovered by positive selection, plating the transformation mixture onto galactose-based agar medium. The pYES2-GFP-BtpB-derived plasmids were isolated from growing clones, amplified in E. coli, verified by restriction analysis and transformed again in yeast cells to verify that they had lost the ability to inhibit yeast cell growth. Mutations were identified on the positive clones by DNA sequencing.

Yeast cellular ATP measurement by luciferase assay

ATP levels were measured using ENLITEN ATP Assay System (Promega) following manufac-turer´s instructions. Yeast cells were cultured in SR for 18 h and then new SG was added for GAL1-driven gene expression to a final OD600 of 0.3 and cultured for 3 h at 30 ˚C. Approximately 1.8x10 7 cells were harvested in 3 mL of culture and then concentrated by centrifugation for 3 min at 2500 rpm at 4 ºC. Pellets were washed with 1 mL PBS at 4 ºC and stored at -80 ºC for further analysis. For ATP extraction, pellets were resuspended with trichloroacetic acid (TCA, 5%, 10 µL) and immediately neutralized using 500 µL of Tris-acetate-EDTA buffer (TAE 1 ×; 40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA, pH 7.75). The samples were centrifuged for 15 sec at 13000 rpm and then 1:100 diluted in more TAE 1×. Ten µL of this solution was mixed with 100 µL of the rL/L reagent provided by the kit and luminescence was measured using OPTOCOMP1 luminometer (MGM instruments). A standard curve for quantification was prepared using the kit´s reagents.

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Brucella TIR proteins deplete host cell NAD +

Yeast cellular NAD + measurement by mass spectrometry

Yeast cells were cultured as stated for the ATP luciferase assay. Approximately 6x10 7 cells were harvested in 10 mL of culture and then concentrated by centrifugation for 3 min at 2500 rpm at 4 ºC. Pellets were washed with 1 mL PBS at 4 ºC and stored at -80 ºC for further analysis.

Our yeast NAD + extraction protocol is a simplified version of the one described by Sporty et al. [53]. Pellets were resuspended in ammonium acetate (600 µL of 50 mM in MS grade water) and approximately 300 µL of 0.5-0.75 mm diameter glass beads (Reesch) were added to the tube. Cells were bead blasted at 5.5 m/s using a FastPrep-24 (MP Biomedicals) for 30 sec twice, allowing a 5 min incubation on ice in between. Supernatant was recovered by perforation of the bead-blasting tube´s base with a red-hot 0.9 x 40 mm needle. The pierced tube was placed inside a capless 1.5 mL microfuge tube and both tubes were centrifuged together for 3 min at 2000 rpm at 4 ºC. This first cell lysate was stored in a new 1.5 microfuge tube on ice. The glass beads in the bead-blasting tube were then washed one more time with 600 µL of a 3:1 v/v mixture of acetonitrile (MS grade) and ammonium acetate (50 mM in MS grade water). The rinsate was then mixed with the first lysate. The mixture was clarified by centrifugation for 3 min at 13000 rpm at 4 ºC and the supernatant was transferred to an ice-cold 1.5 mL microfuge tube. To standardize results, 150 µL of these lysate were kept for protein concentration measurement by Bradford method.

Samples were filtered with a 0.22 µm PTFE fliter (JASCO) and analyzed by liquid chromatography (LC) coupled to a QQQ mass spectrometer equipped with a turbo ion spray source operating in positive ion mode (LCMS 8030,Shimadzu). Chromatographic separation was performed on a Gemini C18 analytical column (50 mm×2.1 mm I.D., 2.7 µm particle size; Poroshell 120 PhenylHexyl). Injection volume was 10 µL. Samples were delivered over 11 min at a flow rate of 0.3 mL/min through the analytical column at 45 ˚C. The mobile phase was composed of A (3% methanol, 10 mM tributylamine, 3 mM acetic acid in water LC grade, 0.1% formic acid in water) and B (methanol). Mobile phase composition began with 0% B and was increased to 45% B in 2 min, to 50% in 5 more minutes and up to 95% in one minute. The mobile phase was then maintained at 95% B for 2 min and followed by re-equilibration with 0% B over the next 2 min, before injection of the next sample. Quantification of NAD + was performed by multiple reactions monitoring (MRM) mode to monitor the parent ion-product ion (m/z) of the analyte. Mass transitions of m/z 662.10 to 540.00 (CE = +16 V) were used for quantification and m/z 662.10 to 407.90 (CE = +30 V) for identification with a dwell-time of 100 ms. The calibration curve was determined by plotting the peak area of the analyte (Y) versus the nominal concentration (X) with least square linear regression. All analyses were made under ISO 9001:2008 quality management system certification.

Brucella infection of HeLa and iBMDM cells

Immortalized Bone Marrow-Derived Macrophages (iBMDM) were obtained as previously described [54]. HeLa cells were seeded at 1×10 5 cells/well for HeLa or 0.8 × 10 5 cells/well for iBMDM (6 -well plates) overnight and tryptic soy broth Brucella cultures inoculated and incubated for 16 h with agitation at 37 ˚C. Cells were inoculated at an MOI of 500, centrifuged at 400 g for 10 min and incubated for a further 1 h at 37 ˚C for HeLa cells and 30 min for iBMDM and RAW cells. Cells were then washed 3 times with media and incubated for 1 h with media containing gentamycin 50 µg/ml and streptomycin 100 µg/ml. After this time media was replaced to reduce the gentamycin concentration to 10 µg/ml streptomycin 20 µg/ ml.

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Brucella TIR proteins deplete host cell NAD +

Total NAD colorimetric assay

Total NAD (NAD + and NADH) was extracted and quantified from cell lysates (from 2 wells of a 6-well plate for each sample) using the NAD + /NADH Colorimetric Assay Kit (Abcam, ab65348) following the manufacturer's instructions. Briefly, the amount of total NAD was calculated from a standard curve (pmol) divided by the sample volume added to the reaction well (µl) and multiplied by the dilution factor.

TEM assay

RAW cells were seeded in a 96 well plate at 1x10 4 cells/well overnight. Cells were then infected with an MOI of 500 by centrifugation at 4 ˚C, 400 g for 5 min and 1 at 37 ˚C 5% CO2. Cells were washed with HBSS containing 2.5 mM probenicid. Then CCF2 mix (as described in the Life Technologies protocol) and probenicid were added to each well, and incubated for 1.5 h at room temperature in the dark. Cells were finally washed with PBS, fixed using 3% PFA and analysed immediately by confocal microscopy (Zeiss LSM800).

Statistical analysis and software

All data sets were tested for normality using Shapiro-Wilkinson test. When a normal distribution was confirmed a One-Way ANOVA test with a Tukey correction was used for statistical comparison of multiple data sets and Students t-test for two sample comparison. For data sets that did not show normality, a Kruskall-Wallis test was applied, with Dunn's correction, or Mann-Whitney U-test for two sample comparison. 3D protein images were generated using PyMOL (Scho ¨dinger), taking advantage of previously published structural data of BtpA (PDB: 4LZP) and RUN1-TIR + NADP + (PDB: 6O0W). (BAB_RS18145). The time-point of 4h was selected to enable a comparison between the wildtype and the ∆virB9 mutant strain, which lacks a functional type 4 secretion system (T4SS).

Supporting information

S1

These experiments were carried out in RAW macrophages, allowing infection of most cells with B. abortus. BAB1_0296 was efficiently translocated into host cells in a manner significantly dependent on the T4SS, in contrast to BAB1_0466 (Figure 20A andB). We should note that a small number of cells infected with the ∆virB9 expressing BAB1_0296 were positive for CCF2 compared to BAB1_0466 (Figure 20B). These first TEM experiments were carried out by a formal postdoc in the lab, Thais Lacerda, prior to my arrival. At 24h post-infection, we could still not detect translocation of BAB1_0466 with this reporter system (Figure 20C). Hence, we can conclude that BAB1_0296 is likely to be a B. abortus effector, and we have named it as NyxA, inspired by Greek mythology. Nyx is the Greek personification of the night, daughter of Chaos, that seemed appropriate to us after remaining in the dark for so long regarding its function. A Brucella genome analysis then identified another gene, BAB1_1101 (BAB_RS21200), encoding for a protein with 82% identity to NyxA, but without a potential CAAX motif (Figure 21). For consistency, we have named this second protein NyxB and that we found was also translocated by B. abortus into host cells, albeit to lower levels than NyxA at both 4 and 24h post-infection (Figure 20A andC). Curiously, the translocation of NyxB was independent of the T4SS (Figure 20A). To confirm that NyxA was indeed translocated across the vacuolar membrane during infection, we created new strains with NyxA fused on its N-terminus with either HA or 4HA epitope tags.

We then infected HeLa cells, a well-characterized model of B. abortus infection nicely suited for microscopy studies. We could not detect any HA-NyxA at any of the time-points analysed.

However, we could observe translocated 4HA-NyxA at 48h post-infection, accumulating in cytosolic structures in the vicinity of multiplying bacteria (Figure 22A). This was also the case for 4HA-NyxB (Figure 22A, bottom panel). Analysis of fluorescence intensity profiles along a defined straight line across the 4HA-positive structures confirmed the majority of the 4HA signal detected does not correspond to intra-vacuolar NyxA or NyxB. In a proportion of infected cells, 4HA-NyxA and NyxB positive structures could be detected in the nucleus (Figure 22B), suggesting these effectors may also be targeting the host nuclei. To determine if these two effectors could interact, we purified them both and carried out pulldown experiments in vitro. We found that His-NyxA could pull-down purified NyxB (Figure 25A). Furthermore, microscale thermophoresis confirmed this interaction and determined a Kd of 482 ± 126 nM (Figure 25B). Similar results were obtained using His-NyxB against NyxA (Figure 26A andB). Together, these results show NyxA and NyxB interact and can accumulate in cytosolic structures as well as the nucleus, suggesting they are likely cycling between these two cellular compartments.

A B

FT W E FT E 15 15 His-NyxA/NyxB empty column/NyxB

NyxA and NyxB enriched nuclear structures are in close association with PMLnuclear bodies

To determine the nature of these nuclear aggregates formed upon ectopic expression of NyxA and NyxB we labelled cells with antibodies recognizing different nuclear bodies (Figure 27A and B). We found the highest level of colocalization (measured by the Pearson Coefficient Correlation PCC) between NyxA and NyxB. Both effectors also extensively co-localize with PMLs unlike nuclear speckles, suggesting closer association with PML-nuclear bodies. As many proteins that accumulate or transit through these nuclear compartments often interact directly with the PML itself, we next carried out a pull-down experiment with either purified His-NyxA or NyxB and incubated with a cellular extract (Figure 27C). No interaction with PML was detected suggesting these Brucella effectors do not interact with PML in the conditions tested. Interactions with endogenous PML were visualized by western blotting (upper blot), and column binding with anti-His (lower blot). Non-bound fractions (F1 and F2), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa). The cell extract is also shown.

NyxA and NyxB do not interact with SUMO in vitro

As all proteins that associate with PML-nuclear bodies are SUMOylated we wondered if this was the case for NyxA and NyxB. We therefore investigated if NyxA and NyxB could be covalently conjugated by SUMO in vitro. These experiments were done by Mariam Taktek, a

Canadian student that I supervised. We purified both NyxA and NyxB, as well as SUMO2 and UBC9, the SUMO ligase and carried our an in vitro SUMOylation assay. As a positive control GST-RanGA1 was used. A single band was detected in the absence of ATP, indicated with a red asterisk at 46.5 KDa, whereas in the presence of ATP a second band was visible (red square) corresponding to the SUMOylated protein (Figure 28). In contrast, no SUMOylated NyxA nor NyxB were detected, which would have an approximative molecular weight of 25 KDa (Figure 28). Only the non-SUMOylated forms are observed in the presence of ATP (blue asterisk).

The Nyx effectors interact with SENP3, which is necessary for efficient B. abortus intracellular multiplication

To identify potential host-interacting partners of the Brucella Nyx effectors, our collaborator Jean-Paul Borg (CRCM, Marseille) performed a yeast two-hybrid screen. One of the main proteins identified was SENP3, and taking into account the nuclear localization of the Nyx effectors, we focused on this potential target. However, we cannot exclude that the other candidates identified are not relevant (Table 4). To confirm the interaction between SENP3 and the Nyx effectors, we attempted to purify the whole SENP3. We were not successful and instead focused on the purification of the Nterminal region of SENP3 that comprised all the yeast two-hybrid hits (SENP37-159). His-V5tagged NyxA or NyxB were able to pull-down SENP37-159, confirming a direct interaction of these effectors with the N-terminus domain of SENP3 (Figure 29B). No unspecific binding to the column was detected (right panel, Figure 29B). We cannot exclude the involvement of other regions of SENP3, but our data show SENP37-159 is sufficient for this interaction.

NoLS catalytic domain

His-NyxB His-NyxA

As SENP3 seems to be the host target of NyxA and NyxB, we next sought to determine its relevance during infection. Amandine Blanco in the lab carried out these experiments. The depletion of SENP3 was efficiently achieved only after 72h treatment with siRNA (Figure 30A and B), after which cells were infected with wild-type B. abortus. We observed a decrease in CFU counts at 48h post-infection following the depletion of SENP3 (Figure 30C). These results

were confirmed by microscopy counts, which showed a reduction in the percentage of cells with more than ten bacteria at 48h post-infection when SENP3 was depleted (Figure 30D) but siRNA for 72h were infected with wild-type B. abortus expressing DSRed and the percentage of cells with either 1 to 5, 6 to 10 or more than 10 bacteria per cell was quantified by microscopy at 48h postinfection. Data correspond to means  95% confidence intervals from 3 independent experiments, with more than 500 cells being counted for each siRNA treatment. A two-way ANOVA with Bonferroni correction was used to compare the bacterial counts obtained in siControl treated cells with siSENP3 depleted cells, for each subgroup (1-5, 6-10 or >10 bacteria/cell).

The NyxB structure defines a novel family of effectors

To gain further insight into the function of NyxA and NyxB and their interaction with SENP3, Laurent Terradot and Virginie Gueguen-Chaignon (PSF) solved the crystal structure of NyxB at 2.5Å (Figure 31). The asymmetric unit of the crystal contains twelve monomers of NyxB but no significant differences were found between them and thus only the structure of subunit A is described hereafter. The NyxB model encompasses residues 17 to C-terminal residue 134. Lack of density for residues 1 to 16 suggest that this part is flexible. NyxB has a mixed - fold with five  strands and six α-helices with a core made of helices 2 to 4 and a small curved β-sheet formed by 3, 4 and 5. The longest helix 4 interacts with 2 and 6 and is connected to the core via a loop containing two short 310 helices designated 3a and 3b (Figure 31A). Helix α1 is loosely associated with the rest of the protein core and is positioned by the preceding and following loops. In particular, a β-hairpin formed by 1 and 2 packs against 5 and anchors 1 to the protein core. Search for structural homologues did not reveal any significant homology and thus make of NyxB structure a prototype for this protein family.

Size exclusion chromatography coupled to multi-angle light scattering (MALS) experiments

indicate that both NyxA and NyxB form dimers (Figure 32). Two putative dimers (dimer 1 and dimer 2) were identified in the asymmetric unit (Figure 32). The association of dimer 1 (chain A and H) buries a total of 530 Å 2 (Figure 32B) while dimer 2 (chain A and J) relies on fewer interactions burying a total 400 Å 2 (Figure 32B). To determine which dimer(s) existed in solution we used size exclusion coupled to small angle X-ray scattering (SEC-SAXS) done by Célia Bergé in Laurent Terradot's lab on NyxB and NyxA proteins. Data indicates that the Rg of NyxB and NyxA are 2.9 and 2.7 nm, with Dmax values of 10.2 nm and 9.6 nm, respectively.

These data are in agreement with a dimeric form of the proteins given that theoretical Rgs for dimer 1 and 2 are 2.4 nm and 2 nm, respectively while Rg of a monomer of NyxB is 1.4 nm.

The higher Rg observed in solution might be due to the disordered region present at the Nterminal portion of each NyxB monomer (residues 1-16) and NyxA (residues 1-12).

Comparison of NyxB experimental SAXS data with theoretical curves obtained with NyxB dimer 1 or dimer 2 indicates that the best fit is obtained with dimer 1 (χ2= 1.3) compared to dimer 2 (χ2= 3.3) (Figure 32C andD). Ab initio modelling using SAXS data confirmed that the envelope obtained fits better the NyxB dimer1. Similar results were obtained using NyxA (Figure 32C). Collectively, X-ray, MALS and SEC SAXS data clearly establish that dimer 1 (Figure 31A) is the conformation of NyxB and NyxA in solution. This NyxB dimer relies on reciprocal

Identification of the Nyx-SENP3 interacting groove

Taking advantage of the structural information of NyxB, we looked for potential interacting sites. Analysis of the NyxB surface revealed an acidic pocket delineated by residues Y66, D80

and E82 within an acidic patch consisting of amino acids D72, E73, Y70, Y86 and Y63, residues that are strictly conserved in NyxA (Figure 33A). In the context of the dimer, these surfaces are juxtaposed to form an extended concave negatively charged area of around 2000 Å 2 (Figure 33A). The inset shows a close-up view of the area with residues' side chains displayed as ball-andsticks and mutated residues (E82,Y66 and D80) coloured in cyan. (B) Pull-down assay with His-NyxA, His-NyxB or the specific catalytic mutants (His-NyxA MAG or His-NyxB MAG ) immobilized on Ni NTA resins that were incubated with a HeLa cell extract. Empty column was used as a control for non-specific binding. Interactions with endogenous SENP3, NPM1 or Histone 3 (H3) were visualized by western blotting using the corresponding antibody, and column binding with anti-His (lower blot). Non-bound fractions (F1 and F2), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa). The cell extract and the different purified Nyx inputs are also shown.

We mutated Y66, D80 and E82 to obtain His-NyxB MAG , where "MAG" stands for mutated acidic groove and Y62, D76 and E78 to obtain His-NyxA MAG . Purified NyxA or NyxB was indeed able to pull-down endogenous SENP3 from a HeLa cell extract confirming their interactions (Figure 33B). A decreased ability for both His-NyxA MAG and His-NyxB MAG to interact with SENP3 was observed (Figure 33B). However, we could only detect a small amount of endogenous SENP3

in the cell extract with our antibody. As it is well established that when SENP3 is pulled-down from a cell extract, its major cellular partner NPM1 can be easily detected by western blotting, we next probed the same membrane with an antibody against NPM1. NPM1 was not pulleddown by His-NyxA MAG and His-NyxB MAG , confirming that this mutation strongly impaired in their ability to bind the complex SENP3-NPM1 (Figure 33B). As a negative control, the membrane was also probed for Histone 3, an abundant nuclear protein that did not bind to NyxA nor NyxB (Figure 33B).

Together these results identify SENP3 as a target of the Brucella Nyx effectors and identify the acidic groove responsible for this interaction in vitro.

The Brucella Nyx effectors induce delocalisation of SENP3

After showing that NyxA and NyxB interact directly with SENP3, a eukaryotic protein mainly found in the nucleoli, we were intrigued by what impact these effectors could have on SENP3

(Figure 5A, top panel). Ectopic expression of HA-tagged NyxA resulted in a marked reduction of endogenous nucleolar SENP3, which instead formed aggregates in the nucleoplasm (Figure 34A andB). As SENP3 redistribution from nucleoli to the nucleoplasm could be due to starvation or mild oxidative stress [268] [257], we ectopically expressed the mutant HA-NyxA MAG , that we know is less able to interact with SENP3. The mutation of the acidic interaction groove impaired delocalisation of SENP3 by NyxA, confirming this effect was due to NyxA's direct interaction with SENP3. Consistently, analysis of the same images for colocalization of SENP3 with HA-NyxA revealed important recruitment, dependent on the acidic groove (Figure 34C). All the cells quantified are shown, with each colour corresponding to an independent experiment and its corresponding mean (N = 4). NyxB was also able to delocalise SENP3 from the nucleoli and recruit SENP3 via direct interaction, implicating both effectors in this phenotype (Figure 34B, C and D). Next, we investigated the prevalence of these phenotypes during infection. We infected HeLa cells with either the wild-type B. abortus strain, the mutant lacking nyxA, or a complemented strain, expressing nyxA from the chromosome under the control of its promoter. Analysis of the level of SENP3 retained in the nucleoli during infection showed a significant lack of nucleoli localization of SENP3 in cells infected with the wild-type B. abortus strain in contrast with the ∆nyxA strain (Figure 35A andB). The wild-type phenotype was partially restored with the complemented strain but not with a ∆nyxA strain expressing nyxA MAG . This mutation impairs interaction with SENP3 in vitro but does not affect effector translocation during infection (Figure 36). This result shows that NyxA's interaction with SENP3 during infection prevents its accumulation in the nucleoli. nyxA, its complemented strain nyxA::Tn7-nyxA or a complementing strain expressing the mutated acidic groove responsible for interaction with SENP3 (nyxA::Tn7-nyxA MAG ). Data are represented and were analysed as in (B). Not all comparisons are shown. All microscopy images displayed have scale bars corresponding to 5 m. In the case of NyxB, we could also observe a statistically significant increase of SENP3 in the nucleoli in cells infected with the ∆nyxB strain compared to cells infected with wild-type B.

abortus, although to a lesser extent than what we observed for ∆nyxA. However, we could not fully complement this phenotype, possibly due to the low sensitivity of this microscopy approach combined with a weaker phenotype (Figure 37A). As expected, a strain lacking both genes encoding for NyxA and NyxB could not mislocalise SENP3 as the wild-type strain.

Representative images of all strains are shown in the (Figure 37B). These phenotypes could not be evaluated in macrophages, as we could not detect SENP3 in bone marrow-derived macrophages with any of the commercial antibodies available. * * Pescadillo (PES1) (Figure 38A), involved in the maturation of the 28S and 5.8S rRNAs and, similarly to SENP3, requires interaction with NPM1 for nucleolar targeting [268]. A slight effect on NPM1 was observed in some B. abortus infected cells (Figure 20B), but to a lower extent than SENP3, suggesting that the bulk of nucleolar NPM1 remains unaffected.

NPM1

wt DSRed NPM1

We next analysed the nucleolar accumulation of the VCP-like AAA-ATPase (NVL), also part of the complex of proteins involved in the maturation of the 28S and 5.8S rRNAs [282].

Unexpectedly, we observed a striking cytoplasmic accumulation of NVL in punctate structures in all B. abortus infected cells rarely visible in non-infected cells (Figure 39A). It is important to note that significant NVL staining was still observed in the nucleoli of infected cells. To determine if the Brucella Nyx effectors induced the formation of these NVL-positive cytoplasmic structures, Amandine Blanco quantified their number in HeLa cells infected either wild-type or a mutant lacking both NyxA and NyxB. She found that cells infected with ∆nyxAnyxB had fewer NVL-positive structures (Figure 39B), suggesting the Nyx effectors contribute to their induction. This difference was not due to different intracellular bacterial numbers as nyx mutant strains replicate as efficiently as the wild-type in HeLa cells (Figure 40).

Strong induction of NVL cytoplasmic punctate accumulation was also observed in immortalized bone marrow-derived macrophages infected with wild-type B. abortus (Figure 39C), in a Nyx-dependent manner (Figure 39D).

These results indicate that induction of NVL accumulation in cytosolic punctate structures

during Brucella infection is dependent on SENP3. We must now complement the mutant phenotypes to confirm these results. It is important to note that depletion of SENP3 alone was not sufficient to induce NVL cytosolic accumulation, suggesting this phenotype is triggered by the infection. Therefore, we wondered it could constitute a generalized cellular response to intracellular bacteria. We was observed with RPL5 labelling (Figure 45) and also when using 3Flag-tagged NyxA (Figure 46). These data overwhelmingly support the idea that these cytosolic Brucella-induced structures contain NVL, RPL5, and both Nyx effectors, at least transiently. We then focused on other cellular processes resulting in cytoplasmic accumulation of nucleolar proteins. Recent reports have described NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) as a nucleo-cytoplasmic shuttling protein that accumulates in the cytoplasm upon starvation [287]. In this context, NUFIP1 acts as a receptor for ribophagy, a specialized autophagy process dedicated to the degradation of ribosomes to generate nutrients and enhance cell survival [288]. We therefore wondered if NUFIP accumulation in the cytosol, a hallmark of ribophagy, could be enhanced in infected cells.

Indeed, cells infected with wild-type B. abortus resulted in an increase in cytosolic NUFIP1 compared to non-infected cells (Figure 47A), with the appearance of vesicular structures.

These were also enriched in NyxA (Figure 47B). However, these structures were negative for LAMP1, suggesting blockage with lysosomes or that they constitute a different NUFIP1-positive compartment (Figure 48).

Materials and Methods

Cells and culture conditions

HeLa and RAW cell lines were obtained from ATCC and were grown in DMEM supplemented with 10% of fetal calf serum. Immortalized bone marrow-derived macrophages [289] from C57BL/6J mice were maintained in DMEM supplemented with 10% FCS and 10% spent medium from L929 cells that supplies MC-CSF. All cells were routinely tested and were mycoplasma free. When indicated cells were starved with HBSS for 8h.

Brucella strains, cultures and infections

B. abortus 2308 was used in this study. Wild-type and derived strains were routinely cultured in liquid tryptic soy broth and agar. 50 μg/ml kanamycin was added for cultures of DSRed and 50 μg/ml ampicillin for strains expressing pBBR1MCS-2 (4HA and 3Flag constructs) or complemented strains (with pUCTminiTn7T_Km). The list of all Brucella strains constructed is included in Supplementary Table 2.

For infections, eukaryotic cells were plated on glass coverslips 18h before infection and seeded at 2x10 4 cell/well and 1x10 5 cells/well for 24 and 6 well plates, respectively. Bacterial cultures were incubated for 16h from isolated colonies in TSB shaking overnight at 37 °C.

Culture optical density was controlled at 600 nm. Bacterial cultures were diluted to obtain a multiplicity of infection (MOI) of 1:1000 in the appropriate cell culture medium. Inoculated cells were centrifuged at 400 x g for 10 min to initiate bacterial-cell contact followed by incubation for 1h at 37°C and 5% CO2 for HeLa cells and 30 min for iBMDMs. Cells were then washed 3 times with DMEM and incubated a further hour with complete media supplemented with gentamycin (50 μg/mL) to kill extracellular bacteria. The gentamycin concentration was then reduced to 10 μg/mL by replacing the media. At the different time points coverslips were fixed for immunostaining. For enumeration of bacterial colony forming units (CFU), cells were lysed in 0.1% Triton for 5 min and a serial dilution plated in tryptic soy agar. in a EcoRV digested suicide vector (pNPTS138) for NyxB. These vectors were then electroporated in B. abortus and transformants selected using the kanamycin resistance cassette of the pNPTS138 vector. The loss of the plasmid concomitant with either deletion or a return to the wild type phenotype was then selected on sucrose, using the sacB counter selection marker also present on the vector. Deletant (∆) strain was confirmed by PCR and sequencing.

The complementing strain was constructed by amplifying either nyxA or nyxB and their corresponding promoter region (500 bp upstream) with the PrimeStar DNA polymerase (Takara). Insert and pUCTminiTn7T_Km [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF] were digested with BamHI and SpeI and ligated overnight. Transformants were selected on kanamycin 50 μg/mL and verified by PCR and sequencing. To obtain the complementing strain the ΔnyxA or ΔnyxB mutants were electroporated with pmini-Tn7-nyxA or -nyxB, respectively, with the helper plasmid pTNS2.

Electroporants were selected with kanamycin 50 μg/mL and verified by PCR.

Eukaryotic expression vectors

The NyxA and NyxB constructs were obtained by cloning in the gateway pDONR TM (Life Technologies) and then cloned in the pENTRY Myc or HA. The NyxA MAG and NyxB MAG constructs were obtained by site-directed mutagenesis. The 4HA-NyxA and NyxB were clone into pBBR-MCS4. All the primers used are listed in Supplementary Table 2.

Transfections and siRNA

All cells were transiently transfected using Fugene® (Promega) overnight, according to manufacturer's instructions. HeLa cells were seeded 18h prior to experiments at 2x10 4 cells/well for 24 well plates. siRNA experiments were done with Lipofectamine® RNAiMAX Reagent (Invitrogen) according the protocol of the manufacturers. Cells were seeded at 1.4x10 4 cells/well. Importantly, siRNA depletion of SENP3 was done by treatment with 0.1µM siRNA every day for 72h. The following sequence was used AAACUCCGUACCAAGGGUUAU. A scrambled siControl was also used. Some cell detachment was observed following the 3 days siRNA treatment.

Legionella infection

Legionella pneumophila strain Paris was cultured in N-( 2 Proteins eluted were separated by SDS-PAGE, transferred to a PVDF membrane, incubated with specific primary antibodies for 1 h and detected with horseradish peroxidase (HRP)conjugated secondary antibodies by using ClarityTM Western ECL Blotting Substrate (Bio-Rad).

Pulldown assays between cell extract and purified Nyx and MAG mutants, see annexe 3 and the nucleolin as well as between the signals of SENP3 and HA-NyxA/NyxB. For each nucleus, the ratio between the mean intensity of the SENP3 signal in the nucleoli and the mean intensity of the SENP3 signal outside the nucleoli is also calculated. The details can be found in the source code and the comments of the macro. In the same way, this software was used to quantify the colocalization between PML and HA-NyxA/NyxB.

For the Brucella infected HeLa cells, a pipeline in the software CellProfilerref3 was used to measure the Pearson correlation coefficient between the signal of SENP3 and the nucleolin, in the nuclei of the cells. The cells were also classified in two classes according to the intensity of the Brucella DSRed signal in the perinuclear area.

TEM1 translocation assay

RAW cells were seeded in a 96 well plates at 1x10 4 cells/well overnight. Cells were then infected with an MOI of 500 by centrifugation at 4 °C, 400 g for 5 min and 1 at 37 °C 5% CO2.

Cells were washed with HBSS containing 2.5 mM probenicid. Then CCF2 mix (as described in the Life Technologies protocol) and probenicid were added to each well, and incubated for 1.5

h at room temperature in the dark. Cells were finally washed with PBS, fixed using 3.2% PFA and analysed immediately by confocal microscopy (Zeiss LSM800).

Thermophoresis

The method is based on the movement of molecules in a micro gradient of temperature, an effect called "thermomophoresis" which varies depending on whether the molecule is free or in interaction (Figure 21). In the initial state, the proteins do not move. Then the solution protein will be heated by an infrared laser. The laser will cause a gradient of temperature ( 2to 3°C). The molecules in solution will then move from hot to cold. Their movement depends on their charge, their size and their hydration layer. The interaction between a molecule and its partner will modify at least one of these parameters. Once the laser is turned off, the proteins will return to their initial state. The phenomenon of thermophoresis is followed by NyxB is used at the following increasing concentrations : 6,67nM, 13,34nM, 26,68nM, 53,3nM, 106,72nM, 213,44nM, 426,88nM, 853,76nM, 1,7µM, 3,4µM, 6,8µM, 13,6µM, 27,2µM, 54,4µM, 108,8µM, 217,6µM.

SUMOylation assay

In 50µl of reaction solution (20mM HEPES pH9, 20mM NaCl, 0.5mM DTT) were added 1.5µg of purified recombinant protein SUMO2 with 1µg of recombinant protein NyxA/NyxB or RanGAP1 (Biomol international #UW9755), 0.2µg of the SAE1/UBA2 heterodimer (Boston Biochem #E-315), 1µg of the purified recombinant protein hUbC9 in the presence or absence of Mg-ATP solution (Boston Biochem #B-20) at 5mM. This reaction solution is incubated for one hour at 37°C and then the reaction is stopped by 10mM of EDTA. 15µL of the reaction solution is used for analysis by cooomassie blue staining.

Yeast two-hybrid

NyxA was cloned into pDBa vector, using the Gateway technology, transformed into MaV203

and used as a bait to screen a human embryonic brain cDNA library (Invitrogen). Media, transactivation test, screening assay and gap repair test were performed as described [299]

[300] [301] [302].

19. Stats All data sets were tested for normality using Shapiro-Wilkinson test. When a normal distribution was confirmed we used a One-Way ANOVA test with a Tukey's correction for multiple comparisons. For two independent variables, a Two-Way ANOVA test was used. For data sets that did not show normality, a Kruskall-Wallis test was applied, with Dunn's correction. All analyses were done using Prism Graph Pad 6. We next sought to observe directly the effectors translocated in the cell by immunofluorescence microscopy. We succeeded with a strain expressing 4HA-NyxA/B, an approach used many times for the observation of numerous effectors in particular in Legionella and Coxiella [303] [304] by increasing the fluorescence signal.

Supplementary

We observed a cytosolic and nuclear distribution of the effectors in transfection. Nevertheless, 4HA-Nyx as opposed to HA-Nyx is much less frequently found to accumulate in the nucleus suggesting that the 4HA tag disturbs this localization. It seems that this is not a direct consequence of the 4HA tag since 4HA-NopA, an effector of Coxiella burnetii, does not present a problem to localize in the nuclei of infected U2OS cells [304]. Consistently, we observed our Nyx effectors during infection using a 4HA tag sporadically localizing in the nucleus.

Nonetheless, it is possible that due to its size a 4HA tag masks Nyx domains involved in the translocation from the cytosol to the nucleus. NLS have not been identified in Nyx, but Victor Cid's team have shown in yeast that the import of these effectors is active and dependent on the alpha-importin encoded by the SRP1 gene in yeast (figure 51), suggesting either that Nyx present NLS that are not identified to date or that they interact with proteins presenting functional NLS allowing them to reach the nucleus.

We believe that both observed localizations (cytosolic and nuclear) are relevant, suggesting that Nyx must either shuttle between cytosol and nucleus or it is a sequential mechanism with a first step in the cytosol before being imported into the nucleus. Further work will be needed to understand the mechanisms and kinetics for the nuclear import of these effectors. We have identified SENP3 as the eukaryotic target of our two effectors. Moreover, the analysis of the crystallographic structure of NyxB allowed us to identify a pocket of interaction. Thus we confirmed this interaction by co-precipitation experiments with purified NyxA and NyxB against the N-terminal domain of SENP3. We were not able to purify the entire protein or the C-terminal domain, so we cannot exclude that the Nyx effectors do not interact with the Cterminal domain of SENP3. It would have been interesting to be able to carry out measurements of affinity constant between Nyx and SENP3 as well as Nyx mag and SENP3, in order to show a decrease or an abolition of the formation of this complex.

The pocket of interaction observed thanks to the crystallographic structure corresponds to a large acidic domain present on Nyx allowing the interaction with an arginine rich domain present on SENP3 at the level of the NoLS domain. It is at the level of this domain that mTOR phosphorylates SENP3, which allows it to translocate into the nucleoli and to interact with NPM1. It is interesting to note that this interaction between Nyx and SENP3 seems to be similar to that between SENP3 and NPM1. Indeed, NPM1 presents an acidic domain involved in the interaction with the arginine-rich motif of SENP3 [268]. Thus, the interaction between SENP3 and Nyx could mask the domain of interaction with NPM1 but this seems unlikely as we are able to pull-down NPM1 with purified Nyx effectors. It is even more likely that Nyx interactions would mask the mTOR phosphorylation residues of SENP3. Indeed, it seems that the phosphorylation of SENP3 is not necessary for the interaction with Nyx, since the interaction with the purified recombinant proteins of Nyx and the N-terminal domain of SENP3 is observed. Thus NyxA/B would interact with SENP3 upstream of mTOR pohsphorylation thus preventing its nucleolar localization.

In order to understand the role of these effectors in the cell we expressed ectopically in Hela cells the Nyx as well as Nyx mag in which the interaction pocket with SENP3 was mutated. We observe that Nyx are able to recruit SENP3, displacing it from its natural nucleoli localization, and recruitment is dependent on the acidic domain identified on the Nyx. The same observations could be made in infection, especially for NyxA.

To date we do not really understand the interest of Brucella to produce two highly similar proteins. We have observed that NyxA always shows a stronger impact on the phenotypes of SENP3 delocalization, in comparison to NyxB. Furthermore, the effects of NyxA and NyxB are not cumulative consistent with their observed interaction in vitro. On possibility is that NyxA by interacting with NyxB could increase its interaction with SENP3 and this would give NyxB a role of chaperone protein for NyxA.

To add to the complexity of these phenotypes, we observed that BspJ ectopically expressed in HeLa cells, was also able to delocalize SENP3, although less than Nyx, however BspJ does not recruit SENP3 (results not shown) unlike both Nyx effectors. In addition, we observed that cells infected with a mutant strain lacking NyxA still have significant SENP3 displaced from nucleoli. These observations suggest that Nyx may act in concert with other Brucella effectors such as BspJ and SerB. Consistent with these results, deletion of NyxA and NyxB alone does not impact Brucella intracellular replication, however we saw that in cells in which we turned off the expression of SENP3 Brucella had a replication defect, highlighting the key role of SENP3 during infection. It would be interesting to observe the impact on the localization of SENP3 and Brucella's ability to replicate when HeLa cells are infected with strains in which the genes encoding all four effectors, NyxA, NyxB, BspJ, and SerB are mutated.

In addition to the delocalization of SENP3 from nucleoli, our study also established that the cytosolic punctiform structures containing translocated Nyx effectors are enriched in NVL, RPL5 and NUFIP1. NVL is an AAA-ATPase that localizes in nucleoli following its interaction with RPL5 and participates in ribosome synthesis [54].

During infection of HeLa cells or BMDMs by

Brucella we observed a relocation of NVL from the nucleus into punctiform structures of the cytosol, partially dependent on NyxA and NyxB. It seems that NyxA/B is not the only one responsible for this delocalization and that something else during the infection allows it, it would be interesting to observe if SerB and BspJ could also be involved in this phenotype.

These quantifications of the cytosolic structures of NVL were made for the wild type strain and the mutant strain lacking the genes encoding nyxA and nyxB. Nevertheless, it would be very important to quantify the cytosolic structures of NVL when cells are infected with nyxA or nyxB mutant Brucella strains that have been complemented with either nyxA/B wild type or nyxA/B mag. This would allow us to confirm that this phenotype is directly dependent on Nyx interaction with SENP3.

During my thesis, we thought that this was the first time that NVL was observed in cytosolic However, cytosolic NVL was only observed under when AMN was over-expressed and authors

did not analyze endogenous NVL in these conditions. It would be interesting to observe whether this role of NVL in endocytic regulation is involved in Brucella infections.

We have also hypothesized that these cytosolic structures could correspond to autophagy.

More generally, autophagy is a ubiquitous degradation mechanism orchestrated by more than 30 specific proteins called ATGs (autophagy-related genes) (Figure 54) [306]. It is a dynamic membrane process that begins with the de novo formation of vacuoles called autophagosomes encompassing cytoplasmic fractions. The degradation of the sequestered material also called the cargo occurs after fusion of the autophagosomes with the lysosomes, in vacuoles called autolysosomes. Autophagy has multiple functions within the cell. It removes damaged or non-functional components and recycles nutrients to maintain homeostasis. In particular, it is a fundamental process in the host's response to infection by pathogens, involved in both innate and adaptive immune responses. This defense mechanism can be countered or used by intracellular microorganisms for their own multiplication. First of all, membrane supply via autophagy is very often beneficial for both bacteria and viruses. Because of their inability to replicate in an acidic environment, these microorganisms block, at least partially, the maturation stage of autophagosomes. Coxiella burnetii for example, which can survive at acidic pH, uses autophagy for optimal development of its replicative vacuole.

Several studies have implicated autophagy at different stages of Brucella trafficking. The YipA protein that localizes at the site where formation of rBCVs is initiated, is necessary for rBCV formation. This process is dependent on Atg9 and WIP1 [307]. In addition, multi-membrane BCVs capable of incorporation of monodansyl cadaverine, a marker of autophagosomes, have been described in epithelial cells [54] [55]. At the late stages of the trafficking, autophagic vacuoles are formed that mediate bacterial exit from infected cells. Current data converge towards a model where there is a very selective hijacking of autophagy components at different stages of BCV maturation, with dependency on Atg9 and WIP1 for rBCVs [307] and ULK and Beclin-1 for aBCVs [START_REF] Starr | Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle[END_REF]. Consistently, inhibition of a large panel of components involved in autophagosome nucleation and elongation did not prevent rBCV formation [START_REF] Starr | Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle[END_REF]. Thus, our study revealed that Brucella-infected cells present NUFIP1 in cytosolic structures corresponding to NyxA/B, NVL and RPL5. NUFIP1 is a receptor for selective autophagy of ribosomes, a nuclear protein that is delocalized in the cytosol when ribophagy is induced. In humans, ribophagy leads to the degradation of the small and large ribosomal subunit. It is triggered by various stresses: starvation, inhibition of mTOR by Torin1, sodium arsenite, oxidative stress such as oxygen peroxide or reversin, an inhibitor of MPS1 that leads to protein imbalance due to poor chromosomal segregation [309]. In human cells the loss of NUFIP1 leads to an inability of the cell to degrade ribosomes following starvation. During the response to starvation NUFIP1 changes location. In the cytoplasm, NUFIP1 will bind to the ribosome and deliver its cargo to the autophagic vesicle by binding to LC3B through its LC3 Interaction Region (LIR) [287].

The presence of the RPL5 ribosomal protein of the large subunit 60S in these cytosolic structures reinforces the idea that we are dealing with ribophagy. We could also label other ribosomal proteins such as RPS3 associated with the 40S subunit or RPL28 which is associated with the 60S subunit and observe by confocal microscopy whether they colocalize with NUFIP1, NyxA/B or NVL. On the other hand, the presence of NVL and NyxA/B are rather surprising since they are not associated with ribosomes. It is interesting to note that NVL has several potential LIR sequences and NyxA/B also has one. Furthermore, it would be interesting to select infected cells expressing 4HA-NyxA/B using a cell sorter and to perform coimmunoprecipitation using an anti-HA resin to purify these NyxA/B /RPL5/NVL/NUFIP1 structures and to perform mass spectrometry in order to identify other associated proteins.

Quantification of the number of NUFIP1 present in the cytosol of cells infected with wild

Brucella and the NyxA/B mutated strain will strengthen our hypothesis that NyxA/B may be ribophagy regulators. However, we do know that NyxA/B are not ribophagy activators since HeLa cells expressing NyxA/B do not present cytosolic NUFIP1. It is possible that Brucella infection causes a kind of starvation that activates the ribophagy. It would be interesting to measure the nutrient status of cells infected with either wild-type or nyxAnyxB mutants.

Starvation-induced ribophagy provokes nucleolar delocalization of SENP3. Recently a study showed that SENP3 regulates autophagy by desumoylating beclin1 (BECN1). Cells not expressing SENP3 show an accumulation of LC3B due to increased autophagosome formation.

BECN1 is required for the initiation of autophagy. BECN1 forms a complex with PIK3C3 important for autophagosome formation. SUMOylation of BECN1 on K380 promotes interaction with PIK3C3 accelerating autophagosome formation. On the other hand, SENP3 deSUMOylate BECN1 which weakens the interaction with PIK3C3 leading to a decrease in the formation of autophagosomes. According to the authors, when starvation occurs, SENP3 accumulates in the cytosol, allowing it to interact with BECN1. As a result, SENP3 can negatively regulate autophaghy [310]. It seems that SENP3 is a kind of a guard to prevent the cell from digesting too much. So far, I have never been able to observe SENP3 in the cytosol.

Perhaps by using their labeling protocol we will be able to observe if SENP3 is also present in the cytosol in the vicinity of NUFIP1 in a NyxA/B dependent manner. Nyx potentially sequester SENP3 in the nucleoplasm and thus prevent the negative regulation of SENP3 on the ribophagy. Thus, SENP3 could be a specific regulator of ribophagy. The fact that the observed structures are LAMP1 negative suggests that fusion with lysosomes does not occur or that the observation at 48h post infection is too early for these structures to be LAMP1 positive, it would be interesting to perform further kinetics. Brucella may also divert ribophagosomes to obtain nutrients and membrane. How can BCV recruit these structures? How can Brucella metabolize ribosomes? Currently we cannot totally exclude that potentially these NUFIP1 positive structures do not correspond to ribophagy, experiments are in progress to confirm this.

During my thesis, I also spent time setting up an iBMDM infection protocol with Brucella, in order to obtain a maximum of infected cells. The objective was then to perform a mass spectrometry SUMOscan to obtain the SUMOyloma of Brucella WT versus Brucella nyxAnyxB infected cells. Unfortunately, I did not have time to perform this experiment, one can imagine that BECN1 would have emerged from this screening, as well as many other proteins.

In conclusion, we have identified two novel Brucella effectors that target SENP3 and induce the relocation of this nucleolar protein during infection. We propose that the SENP3 interaction leads to the induction of ribophagy, visible with the appearance of cytosolic structures enriched in NUFIP1, NVL, RPL5, and the two Nyx Brucella effectors that may allow replicative Brucella-containing vacuoles to obtain nutrients or membranes while promoting cell survival. NyxA and NyxB can act as positive regulators of ribophagy by diverting SENP3, which normally acts as a negative regulator of ribophagy. In addition, our work suggests that NVL is potentially a novel ribophagy marker. In this manuscript we highlighted a role of the S. aureus TIR protein TirS, present on a chromosomal cassette containing multiple antibiotic resistance genes, on the control of virulence. In addition, this was the first study to show upregulation of a TIR protein upon antibiotic treatment and an epidemiological link with enhanced success of bacterial spread.

My contribution was on the evaluation of the intracellular localization of TirS in transfected cells and attempts to express and purify TirS in E. coli for in vitro assays.

Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S.

aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked.

Introduction

The innate immune system constitutes the first line of host defense against invading microbial pathogens in multicellular organisms. Key components of the innate immune response are pattern recognition receptors, which recognize a wide range of conserved bacterial structures, collectively called pathogen-associated molecular pattern and initiate an intracellular signaling immune cascade [1]. The Toll-like receptor/interleukin (IL)-1 receptor (TLR/IL-1R) superfamily, which comprises Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs), is required for many host innate immune responses and characterized by the presence of Toll/ interleukin-1 receptor (TIR) domains cytoplasmically located on each TLR [2]. The TIR domain is critical for protein-protein interactions between TLRs with the corresponding TIRcontaining adaptors. These interactions activate specific transcription factors such as nuclear factor-κB (NF-κB), which regulates the expression of various inflammatory mediators [3,4].

The TIR domain therefore plays a pivotal role in signaling from these receptors, and their importance in immune regulation has made them the subject of intense study.

The TLR signaling pathway is a key target of pathogen mechanisms of host immune system evasion [4]. Indeed, microbes can target various levels of the TLR signaling pathway, from modification of pathogen-associated molecular patterns to modifications in the immune signaling cascade. A potential host evasion mechanism involving TLRs came to light with the identification of bacterial TIR homologues. The majority of studies on bacterial TIR proteins have focused on their potential role as virulence factors that directly subvert host TLR signaling. For example, TIR-like protein A (TlpA) from Salmonella enterica serovar Enteriditis reduces NF-κB activation by a TLR4, IL-1R, and MyD88-dependent pathway and modulates IL-1β secretion during infection [5]. TcpC in the uropathogenic Escherichia coli CFT073 and Btp1/BtpA/TcpB in Brucella species suppress TLR2-and TLR4-mediated activation of NF-κB by targeting MyD88 [6,7]. A second TIR-containing protein in Brucella ssp. (BtpB) was reported to be a potent inhibitor of TLR signaling, probably via MyD88 as well [8].

The presence of a putative TIR-domain-containing protein in Staphylococcus aureus was suggested through a data search analysis [5] before being recently confirmed [9]. S. aureus is an important human pathogen that causes a wide variety of community and healthcare-associated infections [10]. This bacterium has a proven ability to adapt to the selective pressure of antibiotics. S. aureus was initially methicillin-sensitive (MSSA) but isolates resistant to this antibiotic were identified soon after its introduction (MRSA, or methicillin-resistant S. aureus) [11]. S. aureus becomes resistant to methicillin mainly by the acquisition of the methicillinresistant gene mecA, which occurred first in hospital settings and now takes place in the community and in livestock [12,13]. The mecA gene is carried on a particular class of mobile microbial attack. TLR signaling elicits a proinflammatory response that controls immune cell recruitment to infected tissues. Here, we show that Staphylococcus aureus, an opportunistic human pathogen, expresses a host defense-like protein, TirS, that actively perturbs the initial TLR activation stage. Results with isolated human cells and mouse models show that TirS is a broad innate immune inhibitor of TLR-dependent signaling and modulates bacterial virulence, attenuating local inflammation. Moreover, the tirS gene lies near antimicrobial resistance genes for an antibiotic that enhances TirS production, shifting the balance to favor the pathogen and promote disease. Understanding mechanisms by which S. aureus modulates the immune response may lead to novel approaches for preventing and treating infection. genetic elements prevalent in staphylococci, the staphylococcal chromosomal cassette (SCC), designated as SCCmec [14]. Askarian et al. [9] characterized the TIR domain protein TirS in the SCC476 element of the methicillin-susceptible S. aureus strain MSSA476. SCC476 is integrated at the same site on the chromosome as SCCmec elements in MRSA [15]. TirS interferes with the TLR2-induced MAPK and NF-κB signaling pathway and enhances bacterial survival within the host [9].

In the present work, we report that TirS is spread among 12% of MRSA and MSSA strains. In an attempt to describe the genetic context of tirS (for staphylococcal TIR gene) in S. aureus, we fully sequenced the SCC element of representative bacterial strains. In all MRSA and MSSA lineages, the tirS gene was invariably located within this mobile genetic element and co-located with the fusC and mecA (for the MRSA strains) antibiotic resistance genes. Interestingly, our results show that sub-inhibitory concentration of fusidic acid induced overexpression of tirS. We also confirm previous findings that tirS expression induces a negative regulation of the TLR signaling pathway. Our results with a mouse model of skin infection support that TirS modulates bacterial virulence through attenuation of host inflammatory responses during infection. This work is the first description of a TIR homolog protein carried by a mobile genetic element conferring resistance to antibiotics, suggesting a potential selective advantage. Indeed, these features may contribute to the ability of S. aureus to survive and establish a critical population size.

Results

tirS distribution and molecular epidemiology in S. aureus lineages

To assess the prevalence of tirS in various MRSA and MSSA lineages, a series of 226 well-characterized clinical isolates from more than 27 clonal complexes (CCs) or sequence types were subjected to tirS-specific PCR. Among the 226 strains examined, 28 (12.4%) yielded positive tirS amplification (Table 1). tirS was detected in MRSA and MSSA strains belonging to only 3 CCs: CC1, CC5, and CC8. In detail, the tirS gene was detected in 18/18 MRSA strains, CC5 Geraldine clone; 1/6 MRSA strains, CC5 pediatric clone; 1/2 MRSA strains, CC1; 6/9 MSSA strains, CC1; and 2/10 MSSA strains, CC8. Of further interest was our finding of a perfect association between tirS gene amplification and the MRSA Geraldine clone.

We next examined the molecular epidemiology of tirS in human staphylococcal infections. S. aureus strains were isolated from clinical specimens of individuals presenting skin and soft tissue infections, community-acquired pneumonia, bacteremia, infective endocarditis, or nasal colonization (asymptomatic bacterial carriage). No significant association was detected between specific disease and the presence of tirS. Among the 28 tirS-positive clinical samples, 10 were isolated from healthy patients (asymptomatic portage; 36%), 8 from patients with cutaneous infection (29%), 5 from patients with pneumonia (18%), 3 from patients with osteomyelitis (11%), 1 from a patient with bacteremia (4%), and 1 from a patient with infective endocarditis (4%).

Genomic context of the tirS gene in MRSA and MSSA

Because the tirS gene was previously described on a staphylococcal chromosomal cassette SCC476 [9], we performed whole-genome shotgun sequencing of six representative MRSA and MSSA strains positive for the presence of the tirS gene. These included four MRSA lineages: the prototype Geraldine clone strain HT20030749 (CC5; SCCmec I), strain ST20120331 (CC5, SCCmec IV), and strains ST20121850 and ST20130096 (CC1; SCCmec V fusC+) (ENA database project study accession number PRJEB12840; sample accessions ERS1070204 to ERS1070207). Two MSSA strains tirS-positive were also added to the study: strain ST20110167 (CC1) and strain ST20121341 (CC8) (sample accessions ERS1434451 and ERS1434452). As reference to the comparison with our sequenced strains, we used the genome of strain MSSA476, in which tirS was recently described [9]. Whole-genome alignment and search for the site-specific insertion sequences (ISS) typical of SCC-like cassette insertions showed that tirS is invariably present within the SCC element of all six MRSA and MSSA genomes analyzed (Fig 1). Moreover, this gene is found in a highly conserved region within the J1 region (between the ccr complex and the 5' ISS(L)), consisting of five open reading frames (ORFs) that include the tirS gene but also the fusC gene, responsible for resistance to fusidic acid. This region is present as well in MRSA as in the MSSA strains analyzed (Fig 1). Furthermore, BLASTn searches using the tirS and its surrounding four ORFs ("tirS region"), as annotated in the strain MSS476 genome (GenBank: BX571857.1), against the Gen-Bank nucleotide collection (nr/nt) revealed the presence of the tirS region in 14 MRSAs, 2 other MSSAs, and a methicillin-sensitive Staphylococcus hominis strain. Unexpectedly, in all 17 staphylococci, the tirS region was conserved and located in SCC elements in the vicinity of the site-specific recombinase of type ccrAB. For 15 of the S. aureus strains in which the tirS region was detected, a whole-genome shotgun sequence was available, and we observed that they belonged to four distinct clonal complexes: CC1 (2 MRSA and 1 MSSA), CC5 (7 MRSA), CC8 (1 MSSA), and CC22 (4 MRSA).

IV USA300 4 0/4 (0) IV Other 2 0/2 (0) IV MRSA-44 2 0/2 (0) IV USA700 2 0/2 (0) V MRSA-91 1 0/1 (0) CC6 IV WA MRSA 51 2 0/2 (0) CC9 - NA 1 0/1 (0) CC10 - NA 1 0/1 (0) CC12 - NA 3 0/3 (0) CC15 - NA 5 0/5 (0) CC20 - NA 2 0/2 (0) CC22 - NA 8 0/8 (0) IV EMRSA-15/Barnim/Middle Eastern 5 0/5 (0) CC30 - NA 20 0/20 (0) IV Southwest pacific 3 0/3 (0) ST34 - NA 4 0/4 (0) ST36 II EMRSA-16 2 0/2 (0) CC45 - NA 8 0/8 (0) IV Berlin 2 0/2 (0) IV Other 2 0/2 (0) CC59 - NA 3 0/3 (0) IV USA1000 2 0/2 (0) V Taiwan 2 0/2 (0) V Other 1 0/1 (0) CC80 IV European 4 0/4 (0) CC88 IV WA MRSA-2 2 0/2 (0) V Other 2 0/2 (0) ST93 IV Queensland Clone 2 0/2 (0) CC97 - NA 5 0/5 (0) (Continued )

TirS production

To investigate TirS production by S. aureus, tirS expression was examined by quantitative realtime reverse transcription PCR (RT-qPCR) during bacterial growth in one MRSA (clone Geraldine, CC5) and one MSSA (CC1) tirS-positive strains. The analysis of the tirS expression kinetics showed stable expression of tirS during growth with a marginal increase at the end of the exponential phase (Fig 2A). The difference in tirS expression and the standardization gene hu was generally around seven cycles.

Because tirS is located in the SCC element, we investigated the effect on tirS expression of adding a sub-minimum inhibitory concentration (sub-MIC) of oxacillin (1/2 MIC) and fusidic acid (1/4 MIC) during the exponential phase of S. aureus growth in 9 strains. Addition of fusidic acid appeared to significantly modulate tirS expression both in MSSA and MRSA strains For all 6 strains, the tirS gene was found within the SCC element. Schematic diagrams represent the genomic comparison of the 6 SCC regions of both MRSA and MSSA strains used in our study and the SCC476 element of strain MSSA476 for which the tirS gene was first described [9]. Predicted ORFs are marked in the direction of transcription as arrows. The highly conserved tirS region, consisting of the tirS gene and 4 surrounding genes among which we find the fusC gene (fusidic acid resistance), is represented by green arrows. Light rose arrows represent genes coding for antibiotic resistance, orange arrows represent the mecA gene conferring resistance to beta-lactams and blue-gray arrows represent the site specific recombinases (ccrAB or ccrC) of SCC elements. The SCC elements ISS(L) and ISS(L)) are also represented as black vertical bars. Abbreviations: ccrAB = cassette chromosome recombinase A and B; fusC = fusidic acid resistance gene; mecA = methicillin resistance gene; ccrC: cassette chromosome recombinase C; ble = bleomycin resistance gene; aaD/knt = kanamycin nucleotidyltransferase (resistance gene). doi:10.1371/journal.ppat.1006092.g001 (Fig 2B). tirS was upregulated up to six-fold with fusidic acid compared to its expression without antibiotic stress and level of induction by fusidic acid was strain dependent. In contrast, no difference in levels of expression of tirS was observed with exposure to sub-MIC of oxacillin.

TirS interferes with TLR signaling

TirS belongs to the family of bacterial TLRs [9]. We therefore investigated the ability of TirS to specifically interfere with TLR signaling using an in vitro NF-κB-dependent luciferase reporter system. Although a variety of TLR receptors have been described, in humans the most relevant for recognition of bacterial molecules are TLR2, TLR4, TLR5, and TLR9. Ectopic expression of of antibiotic treatment. Bacterial strains without stress were used as reference (relative quantity = 1) to estimate the relative quantity of tirS mRNA. White bars correspond to negative control (no antibiotic), grey bars to bacteria exposed to oxacillin, and black bars to bacteria exposed to fusidic acid. Data represent mean ± SEM of three to six independent assays. * p < 0.05; ** p < 0.01; *** p < 0.001. doi:10.1371/journal.ppat.1006092.g002 tirS in HEK293T cells transfected with the luciferase reporter vector and various TLRs resulted in reduced TLR2 activation and to a lesser extent, reduced activation of TLR9, TLR4, and TLR5 (Fig 3A). As a control, we confirmed that this inhibitory effect of TirS on murine TLR2 was dose-dependent by carrying out the transfections with increasing amounts of the expression vector encoding TirS (Fig 3B). Inhibition of human TLR2 and TLR4 pathways following addition of the appropriate ligands was also observed (Fig 3C). These results suggest that TirS may, at least partly, target a common molecule between these pathways such as the adaptor molecule MyD88. Consistently, reduction of IL-1R following IL-1β stimulation was also observed in the presence of TirS (Fig 3D). In contrast, although much lower levels of activation can be obtained for TLR3, which is independent of MyD88, we did not observe any significant effect of TirS on this pathway (Fig 3E). This was also the case for endogenous TNF receptor (TNFR) (Fig 3D). Overall, these results are in agreement with data from Askarian et al. [9] that previously described TirS inhibition of TLR2, MyD88 and TIRAP dependent pathways in vitro.

In addition to its ability to interfere with TLR signaling, the bacterial TIR effector protein from Brucella BtpA targets and modulates microtubules [16] through a WxxxE motif [17] as well as the recently identified TIR protein from Bacillus anthracis referred to as BaTcp [18]. Since the WxxxE is also present in TirS, we investigated the intracellular localization of ectopically expressed TirS by confocal microscopy on HeLa cells transfected with myc-TirS and indirect immuno-fluorescence staining with anti-myc antibodies. We found that myc-tagged TirS accumulated in filament-like structures of irregular shapes within the host cytosol (Fig 3F). Similar results were observed for GFP-TirS, suggesting that this localization was not dependent on the tag (S1 Fig) . In addition, TirS showed no co-localization with cytoskeleton components as observed after labeling with either phalloidin for actin, tubulin for microtubules, or vimentin for intermediate filaments (Fig 3F), suggesting a different targeting than previously described for BtpA and BaTdp.

TirS modulates S. aureus virulence

To further investigate the role of TirS during infection, we carried out in vivo studies in mice. Because S. aureus is the leading cause of skin infection, we developed a subcutaneous model of infection in mice with the inoculation of S. aureus clone Geraldine-wild type (WT) strain, a deleted for the tirS gene (ΔtirS) strain, or a ΔtirS strain restored for tirS gene in a chromosomic position (ΔtirS + tirS). First, we confirmed that bacterial growth was not affected by genetic manipulation of TirS by assessing growth in two different media (brain-heart infusion (BHI), tryptic soy broth (TSB)) (S2 Fig) . Then, wild-type (WT) C57Bl/6 mice were inoculated with the three strains. Cutaneous infection resulted in the development of visible lesions by day 1 that healed by day 14 regardless of the strain (Fig 4A and4B). As control, injection of sterile phosphate buffered saline (PBS) did not induce any skin lesions in mice. Infection with ΔtirS strain resulted in larger lesions (~2.5-fold) compared with the WT strain (p < 0.01). These differences appeared around day 6 and persisted for about 4 days before being resolved at the same time for both strains. Similar lesion sizes were observed in mice infected with the S. aureus restored strain compared with the WT strain (p = 0.9), confirming the role of TirS on the observed results. To better understand the factors that might be important in determining the size of skin lesions in the mouse model, animals were sacrificed between 5 and 9 days after infection induced by MRSA clone Geraldine WT and ΔtirS strains to the sample lesion. First, we enumerated the bacterial load in the skin lesion. No significant differences in colony-forming unit (CFU) counts between the strains were observed (Fig 5A). Moreover, lesion size was not correlated with bacterial burden in either the WT or Δ-tirS strain (S. aureus WT: r 2 = 0.39; p = 0.2, S. aureus ΔtirS: r 2 = 0.35; p = 0.2) (Fig 5B). This finding raised the possibility that the inflammatory response was more important in determining lesion severity, as assessed by lesion size, than bacterial burden in the lesions. To follow up on these observations, we sought to measure neutrophil and macrophage activity with the quantification of levels of myeloperoxidase (MPO) and a panel of inflammatory cytokines in the skin lesions of mice infected with MRSA clone Geraldine WT and ΔtirS strains. Interestingly, the levels of MPO and IL-1β, IL-6, IL-17, CXCL1, and MCP1 were significantly lower in the murine skin lesions developed after the WT strain inoculation compared with the ΔtirS strain (Fig 5B). Correlation between skin lesion size and the levels of MPO, IL-1β, IL-6, IL-17, CXCL1, and MCP1 was statistically confirmed for each of these inflammatory markers (MPO: r 2 = 0.71, p < 0.001; CXL1: r 2 = 0.55, p < 0.001; IL-1β: r 2 = 0.61, p < 0.001; IL-17A: r 2 = 0.45, p < 0.01; MCP1: r 2 = 0.74, p < 0.001; IL-6: r 2 = 0.64, p < 0.001) (Fig 5C). Therefore, the smaller lesions observed after experimental inoculation with the bacterial WT strain were associated with lower levels of chemokines and less inflammation but not with decreased bacterial burden. These data support the notion that the local inflammatory response is a key determinant of lesion size. These results are consistent with a role for TirS in the control of the inflammatory response during S. aureus infection in vivo.

Finally, to evaluate the contribution of the TIR-containing adaptor protein MyD88, a key component of the TLR signaling pathway implicated in proinflammatory mechanisms [19], MyD88-deficient mice from the C57BL/6 genetic background were subcutaneously inoculated with MRSA clone Geraldine WT and ΔtirS strains. An identical infection protocol and bacterial concentration were used as in WT mice. As control, injection of sterile PBS did not induce any skin lesions in mice. Interestingly, there was no significant difference in lesion size between MyD88-deficient mice infected with the WT or ΔtirS S. aureus strain (Fig 6), suggesting that MyD88 might play a role in virulence of the MRSA clone Geraldine used. These results confirm in vitro previously reported data showing that TirS inhibits signaling in a MyD88-dependent manner [9].

Discussion

Bacterial strategies for innate immune evasion involve manipulation of the TLR signaling by TIR homologues such as TirS for S. aureus [20]. In this work we report the localization of the tirS in different SCC elements and its role in the control of the inflammatory response during S. aureus infection. Using a mouse model of S. aureus skin infection, we evaluated the role of TirS on S. aureus virulence. We show that S. aureus Geraldine deleted for the tirS gene exhibited superior virulence compared to the WT strain, as attested by the size of the skin lesion. Of note, bacterial counts in the skin lesions did not differ between the mutant and the WT strains and did not correlate with clinical severity (i.e., lesion size). This finding suggests that bacterial burden may not be the primary driver of lesion severity and argues that lesion severity may be due, at least in part, to the associated inflammatory response. In support of this hypothesis, we found a correlation between lesion size and levels of the proinflammatory cytokines, as well as neutrophil activity (as assessed by MPO levels). These findings are consistent with previous studies, which underscores that the severity of skin infection is often driven by the inflammatory response to the invading pathogen as much or more than by the direct effects of the pathogen itself [21][22][23]. This inference suggests that the attenuation of skin inflammation observed with S. aureus WT strain, compared to its tirS mutant, was driven by modulation of the inflammation resulting from TirS action. Such a conclusion is concordant with the fact that the ectopic expression of TirS in eukaryotic cells appeared to temper stimuliinduced TLR2-, TLR4-, TLR5-, and TLR9-mediated NF-kB activation. Accordingly, a previous and independent work has reported a negative interference of TirS with the TLR2 signaling pathway [9]. These results can be directly linked to our in vivo observations in mice, explaining the modulation of virulence during S. aureus infection by tirS [4,20]. Here the bacterial TIR effector has been shown to induce attenuation of virulence during infection because of the downregulation of the innate immune pathway.

In addition to control of inflammatory responses, some TIR domain-containing proteins such as BtpA or BaTcp have been shown to target and modulate microtubules [18,24] through a WxxxE motif that is also present in TirS and involved in cross-talk between the TLR and GTPase signaling pathways [16]. We found that ectopically expressed TirS accumulated in filament-like structures of irregular shapes within the host cytosol but that do not co-localize with typical cytoskeleton components (microtubules, actin or intermediate filaments). This observation is different from those previously described for other bacterial TIR proteins from Brucella [8,16] and BaTdp from B. anthracis [18]. The role of TirS accumulation in filament-like structures in the modulation of S. aureus pathogenicity remains to be explored.

The host interacting partner of TirS has yet to be identified. MyD88 is a general adaptor protein that plays an important role in Toll/IL-1 receptor family signaling. In vitro results of Askarian et al. [9] showed that TirS interferes with TLR2 and both MyD88 and TIRAP pathways in vitro. Here we report that in vitro, TirS is a potent inhibitor of not only TLR2 but also TLR4, TLR5, and TLR9. All of these proteins are dependent on MyD88, a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signaling, which argues for an interaction of TirS with MyD88. Consistently, we found that S. aureus carrying tirS induced no increased virulence in a MyD88 knockout mice model. Taken together, these results suggest that TirS modulates an inflammatory response at the site of the infection through the MyD88 adaptor protein. Preliminary work from our lab has not been able to purify TirS, which is highly insoluble and failed to detect an interaction by co-immunoprecipitation assays. Further work is now required to understand the molecular mechanism by which TirS interacts and/or competes with MyD88.

As yet, there is no clear consensus on the mechanism by which TIR proteins enter host cells and localize to the host cell cytoplasm. From deduced amino acid sequences, no recognizable signal sequence for secretion was detected in TIR proteins, including TirS. In the case of TlpA from Salmonella enterica, for which no direct evidence of secretion has been reported, the suggested mechanism is a role for type III or type IV secretion systems (T3SS or T4SS) that can directly inject TIR effectors into the host cells [5]. TcpC of E. coli has been reported to be secreted into the media of cultured bacterial cells and subsequently taken up into host macrophages to interfere with TLR-mediated tumor necrosis factor induction [17]. In the case of Brucella, BtpA and BtpB are translocated into host cells in a manner partially dependent on the T4SS [8]. As observed with other TIR protein genes, the tirS gene is not co-located with genes encoding for a secretion system in S. aureus. However, TirS has been reported to be secreted into the media through an unknown mechanism [9]. Further studies are needed to clarify this issue, perhaps by performing real-time imaging and co-localization studies.

A comparison of different SCC elements in various S. aureus genetic backgrounds highlights the invariable presence of tirS within the SCC fusC/tirS mobile genetic element, sometimes included in the SCCmec elements in the J1 region. As proposed previously [9,25], the finding that tirS region was invariably present within an SCC element suggests that similar to mecA and fusC transmission, SCC-mediated horizontal transfer is the major mechanism of tirS dissemination. Moreover, horizontal transfer of the tirS region is also suggested with the nearby presence of site-specific recombinases of the ccrAB type in all SCC elements carrying the tirS gene [25]. Of further interest, all published sequences of SCC fusC/tirS have identified four conserved additional genes. Future work should attempt to assign functional roles of these putative proteins and evaluate their involvement in the regulation or modulation of tirS and fusC transcription.

The co-location of mecA and fusC genes in this genetic element suggested that, in addition to antibiotic resistance genes, the presence of tirS may also confer a selective advantage for S. aureus. However, our observation indicated that a sub-inhibitory concentration of fusidic acid but not oxacillin induced overexpression of tirS in all strains tested. These results were unexpected because we observed the opposite outcome for Panton Valentine leukocidine and alpha-toxin expression with the same antibiotics: overexpression by oxacillin and inhibition by fusidic acid [26]. The acquisition by S. aureus of a gene encoding for factors that modulate virulence in the SCCmec element is not exceptional. The pls gene encoding for a large surface protein with an LPXTG peptidoglycan-anchoring sequence is a part of the type I SCCmec element [27]. This protein reduces S. aureus adherence and invasiveness [28]. Pls was also described in SCCmec III [29] but never in MSSA. Conversely, genes encoding for phenol soluble modulins alpha 1 to 3 (PSM-α1-3; small peptides with an amphipathic α-helical structure and strong surfactant-like properties that induce the production of proinflammatory cytokines and recruit, activate and lyse neutrophils) were first described in MSSA [30] before being found to be part of SCCmecII, SCCmecIII, and SCCmecVIII (PSM-α-mec) [31,32]. To our knowledge, is

it not yet known whether antibiotics whose resistance is encoded by these SCC elements modulate the expression of Pls and/or PSM-mec.

A number of bacteria expressing TIR-containing proteins have been described, but as far as we know, only one study references their prevalence throughout a bacterial species [24]. In this work, we report the presence of tirS gene in 12.4% of a clinical MSSA and MRSA S. aureus strain collection. The TirS effector was not shown to be associated with a specific clinical human presentation by molecular epidemiology studies. For the Geraldine clone that always holds the tirS gene, previous observation did not identify a particular association with disease severity [33,34]. By contrast, the first nosocomial outbreak due to the Geraldine clone was recently reported, emphasizing its efficiency in being transmitted and easily spread within health care settings [35]. Indeed, this clone has been reported to be both a communityacquired and hospital-acquired MRSA. Thus, the clone Geraldine SCCmec element may provide a selective advantage in both settings: in the hospital because of a higher antimicrobial resistance compared to drug-susceptible WT S. aureus strains, and in the community because of its enhanced inhibition of the innate immune response. Similarly, different groups recently described the emergence in the community and in the hospital of ST1, ST45, and ST149 MRSA fusC positive strains in England and of fusC positive ST5 MRSA in New Zealand that also are tirS positive [36,37]. These epidemiological observations highlight the selective advantage of S. aureus in carrying the SCCmec element with SCC fusC/tirS.

In summary, we identify for the first time a bacterial TIR homolog protein genetically linked to an antimicrobial agent resistance determinant in the genetic mobile element SCC, thus providing a molecular connection between two key traits determining the successful outcome and spread of bacterial infections. Moreover, TIR homolog protein production was modulated by one antibiotic, fusidic acid, for which the resistance is encoded in a conserved region, which includes the tirS gene, and located within these SCCmec and non-mec SCC elements. This result expands knowledge about bacterial TIR homologs that constitute an ingenious strategy of pathogenic bacteria to evade the host immune system. The current state of knowledge strongly suggests that TIR effectors should be considered as potential key effectors of host defense, which emphasizes that further research is required to elucidate the precise mechanism of action of these interesting molecules. From the clinical point of view, the identification of the critical role of TirS signaling for modulating the immune response to a site of infection raises the possibility that this pathway could be locally targeted to engage the host's own immune responses in the treatment of a microbial infection.

Materials and Methods

Ethical statements

Isolates were obtained as part of routine diagnostic testing and were analyzed anonymously. All data were collected in accordance with the European Parliament and Council decision for the epidemiological surveillance and control of communicable disease in the European community [38,39]. Ethical approval and informed consent were not required.

Bacterial strain characterization

A subset of 226 strains from the collection of the Centre National de Re ´fe ´rence des Staphylocoques (Lyon, France), composed of 103 strains of the main community and hospitalacquired MRSA clones, and 123 strains of MSSA were used in this study. They were sent to the laboratory for detection of toxin production in the context of nasal colonization, skin and soft tissue infection, pneumonia, bacteremia, or endocarditis. The S. aureus HT20030749 strain belonging to the clone Geraldine was isolated from blood culture of patient with bone-joint infection.

All strains were genotyped as previously described. Briefly, bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits (Qiagen). The diagnostic DNA microarrays, identibac S. aureus Genotyping (Alere) used for this study, as well as related procedures and protocols, have been previously described in detail [40]. This microarray covers 332 different target sequences corresponding to approximately 185 distinct genes and their allelic variants. The assigning of isolates to CCs was determined by the comparison of hybridization profiles with those previously characterized by using multilocus sequence typing reference strains [40].

Illumina Sequencing. Genomic DNA were extracted from each isolate using a QIAcube extraction kit (Qiagen). The Nextera XT DNA preparation kit (Illumina) was used to generate sequencing libraries from 1 ng of DNA. Whole-genome sequencing was finally done with an Illumina HiSeq (Illumina, San Diego, CA, USA) to generate 150-bp paired-end reads.

De novo assembly. For each isolate, the raw paired-end reads were assembled using a modified version of the A5-miseq open-source pipeline [41]. This pipeline implements a complete sequencing data processing workflow from raw read cleaning to de novo assembly. The first task of the read cleaning involves removing the regions of the raw reads that are contaminated by the adapters during the Nextera XT protocol using the Trimmomatic program [42]. Then, the reads are filtered and trimmed according to quality and length criteria using Trimmomatic and the preprocess function of String Graph assembler (SGA) [43]. Finally, the correct function of SGA is used to correct errors in the reads by a k-mer frequency-based method. After being quality filtered and error corrected, the reads were assembled by the IDBA-UD500 program, which implements an iterative De Bruijn graph built with several values of k-mer lengths, from low to high values, instead of a single value as for most de novo assemblers [44]. The reads are then mapped against the assembly using BWA-MEM [45] to polish the contigs at every position where basecalls differ between the mapping and the assembly. The scaffolding implemented at the end of the original A5-miseq pipeline was not performed because it provides no gain in the subsequent analysis of marker detection.

Characterization of SCCmec V and tirS regions. First, the three MRSA genomes were aligned against the strain MSSA476 genome using progressiveMauve with default parameters [46]. Then, identification of SCC was done by searching for the ISS of these elements [47,48], both at the end of the ORF X / rlmH gene and further downstream. The newly detected SCCmec elements were annotated using the RAST Server (http://rast.nmpdr.org/), and blastn searches were performed (http://blast.ncbi.nlm.nih.gov/) against the nucleotide collection (nr/nt) using the megablastn algorithm and the organism filter for S. aureus (taxid:1280).

tirS amplification

Oligonucleotide primers tirS-For-CTTCAAAAAGAGCAGTCTAGG and tirS-Rev-CTTCAA CACTCACTTTATGCC were designed according to tirS sequence. After amplification for 30 cycles (30 s of denaturation at 94˚C, 30 s of annealing at 53˚C, and 30 s of extension at 72˚C), the PCR products were resolved by electrophoresis through 1.5% agarose gels (Sigma, Saint Quentin Fallavier, France). This step was followed by SYBR Safe DNA (Life Technologies) staining and analysis. To assess the specificity of tirS amplification, PCR products were subjected to DNA sequencing (Biofidal, Lyon, France). S. aureus HT20030749 and RN4220 strains were used as positive and negative amplification controls, respectively.

Transcription of tirS

tirS expression was analyzed using RT-qPCR. MRSA ST20121850 (CC1), MSSA ST20130407, ST20110167, ST20080979 (CC1), MSSA ST20121341 (CC8), MRSA ST20120331 (CC5, type IV), MRSA ST20111318, ST20110610, HT20030749 (CC5, type V) were grown in fresh MHB at 37˚C, after a 1:100 dilution of overnight cultures. For kinetics analysis, total RNA of two representative strains (MRSA HT20030749, CC5 and MSSA ST20130407, CC1) was purified at 2, 4, 6, and 8 h of growth as previously described [49]. Total RNA was extracted with the RNeasy Plus (Qiagen) kit including a gDNA eliminator column and an additional DNAse treatment (Qiagen). RNA quality and quantity were determined by Bioanalyzer (Agilent) using the RNA Nano chips and quantified using the ND-8000 (NanoDrop Technologies). Absence of DNA contamination was checked by using tirS-specific primers and probe at optimal concentrations (assessed as previously described [50] without the reverse-transcription step). The final concentrations were 0.2 μM for primers and probe (tirS-F-CTATTTGGCATAAAGTGAGTGTT GAAG, tirS-R-AAATCACTTGTATTCAATGCATACTTATCT, and tirS-P-CGTGCATAC AACCCATAT labeled with NED at the 5' and 3'-minor groove binder), and reactions were performed in a one-step RT-PCR enzymatic mixture (Agilent Technologies, Brilliant II QRT-PCR Master Mix Kit) in a final volume of 20 μl in the CFX96 system (Bio-Rad) and following manufacturer's instructions. Differences in Ct values between tested transcripts and hu signals were used for normalization purposes and based on the MHB medium condition at 2 h as a reference. The fold change was expressed as the inverse exponential of the difference between MHB Ct (reference) and the stress condition Ct. This assay was also used on 2-h cultures to assess gene expression values of tirS in various stress conditions such as the presence of 1/2 MIC of oxacillin for MSSA and 5 μg/ml for MRSA or 1/4 MIC of fusidic acid for MSSA and MRSA [49] on total RNA purified after 30 min of exposition. MICs were determined using CLSI recommendations [50], and the stress experiments were performed with drug concentrations showing minor impact on growth rate following control experiments (S3 Fig) .   Construction of the tirS-deleted mutant S. aureus RN4220 (lab strain collection) was used for plasmid amplification and genetic manipulations because it is a nitroso-guanidine-induced mutant capable of accepting E. coli DNA [51]. To delete the tirS gene, we performed allelic replacement using double crossover recombination as previously described [52]. Using the primers and restriction enzymes listed in S1 Table, we generated two fragments of 930 bp 5 0 and 1029 bp 3 0 of tirS. These fragments were ligated in 5 0 and 3 0 of the chloramphenicol acetyl transferase gene [53] and inserted into pMAD [54]. Plasmid and inserts were checked by PCR and sequencing using the primers listed in S1 Table . Plasmid was introduced by electroporation in RN4220 and then in HT20030749 (Geraldine strain). We performed double crossover recombination yielding to deletion of tirS in HT20030749 (Geraldine strain). The mutant strain obtained was called ΔtirS. Gene deletion was checked by PCR and sequencing using specific primer hybridizing with internal and external positions of the deleted region (S1 Table ). Insert presence was checked with primers IngDNA_F and vG_CDS_1_R or IngDNA _F and vG_cat_1_R, yielding a negative PCR and a 2017-bp amplicon for the correct construct.

Construction of the tirS restored strain

Total DNA and plasmid DNA were prepared with Qiagen kits (QIAamp DNA Mini Kit and QIAprep Spin Miniprep Kit) after lysostaphin lyses for S. aureus. When necessary, transformation of E. coli DH5α (Promega, Madison, USA) was performed by treatment with CaCl2, and S. aureus strains were transformed by electroporation (Bio-Rad gene pulser). The tirS-deleted strain was complemented by inserting the tirS gene sequence downstream from the leukocidin promoter P-lukS in the bacterial chromosome using sequences homologous to sequence NWMN_0029 and NWMN_0030 of Newman strain (GenBank: AP009351.1) for chromosomal recombination. The region of NWMN_0029 and NWMN_0030 was amplified using primers New29-523 and New30-2371, restricted by EcoRI and SalI and cloned on a pBluescript vector (Stratagene) to obtain plasmid pLUG37. The tirS gene sequence was amplified and cloned between the P-lukS promoter region and lukF-PV transcriptional terminator of the Panton-Valentine leukocidin genes (respectively amplified using primers phi259/phi748 and phi2648/phi2819) on pBluescript. The whole DNA fragment obtained by SmaI was cloned in pLUG37 in the natural EcoRV restriction site between the NWMN39 and NWMN30 sequences. From the resulting plasmid, the DNA fragment corresponding to NMWN30-PlukS-tirS-term lukF-NWMN29 obtained by EcoRI-SalI restriction was cloned in the pMAD vector (pLUG1158). The resulting chromosomic restored strain was called ΔtirS + tirS. Expression of tirS in the restored strain was confirmed by RT-PCR. Oligonucleotides primers for PCR and DNA cloned subfragments are detailed in S2 Table.

Luciferase activity assay

HEK293T cells (American Type Culture Collection (ATCC), USA) were transiently transfected using Fugene (Roche) for 24 h, according to the manufacturer's instructions, for a total of 0.4 μg of DNA consisting of 50 ng TLR plasmids, 200 ng of pBIIXLuc, a reporter plasmid containing luciferase under the control of two Igκ-κB sites [55], 5 ng of control Renilla luciferase (pRL-null, Promega), and 50 ng of myc-TirS expression vector, unless stated otherwise. In the case of TLR4, MD2 was co-transfected for efficient detection of LPS. When indicated, an increasing amount of vector (ng) was used for the transfections to obtained different levels of expression of TirS. In all cases, the total amount of DNA was kept constant by adding empty vector. Negative control corresponds to empty vector alone (pCDNA3.1). Where indicated, cells were treated with E. coli LPS (1 μg/ml), Pam2CSK4 (100 ng/ml), CpG ODN1826 (1 μM), and Flagellin Fl-ST (1 μg/ml), all obtained from Invivogen, for 6 h, and then cells were lysed and luciferase activity measured using the Dual-Glo Luciferase Assay System (Promega). In the case of IL-1R and TNFR, endogenous detection was monitored following 6 h of stimulation with IL-1β (100 ng/ml) or TNF-α (100 ng/ml). The tirS construct was obtained with the following primers tirS-fw-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTCAGTATTA GAAACTAAATTAAAAAG and tirS-rev-GGGGACCACTTTGTACAAGAAAGCTGGGTCC TAATTCTTAGAATTAACGATTACTTG and then cloned in the gateway (Life Technologies) entry vector and subsequently in the pDEST-Myc (Life Technologies) to create an N-terminal myc tag fusion with TirS.

Immunofluorescence microscopy

HeLa cells (ATCC, USA) were transfected with myc-tirS using Fugene (following manufacturer's instructions) for 10 h. Cells were either fixed in 3% paraformaldehyde, pH 7.4, at room temperature for 15 min or placed in ice-cold methanol for 5 min. Cells were then permeabilized for 10 min with 0.1% saponin in PBS, followed by blocking for 1 h with 2% bovine serum albumin and 10% horse serum in PBS with 0.1% saponin. Primary antibodies were incubated for 1 h followed by three washes in PBS, 1 h incubation for secondary antibodies, two washes in PBS, and one wash in water before being mounted with Prolong Gold. Primary antibodies used were rabbit anti-myc (Abcam) at 1/1000, with either mouse anti-beta tubulin (TUB 2.1) at 1/250 or mouse anti-vimentin (V9) at 1/100 (both Sigma). Secondary antibodies used: donkey anti-rabbit Alexa 488, donkey anti-mouse Alexa 555 (Life Technologies) at 1/1000. The actin cytoskeleton was labeled with phalloidin 568 (Life Technologies). Samples were examined on a Zeiss 710 laser scanning confocal microscope for image acquisition. Images of 1024 × 1024 pixels were then assembled using ImageJ and Adobe Photoshop 7.0.

Growth curves of S. aureus strains S. aureus Geraldine WT and ΔtirS strains were grown in fresh BHI medium or TSB medium at 35˚C, after dilution of overnight cultures to OD600 = 0.03. A thermostated microplate reader (TECAN M200 Infinite Pro) was used to follow bacterial growth by measuring OD600 every 15 min for 24 h. As controls, specific wells were inoculated with medium only. Experiments were done in triplicate.

Murine model of S. aureus subcutaneous infection

Bacterial isolates and growth. S. aureus Geraldine WT, ΔtirS, and ΔtirS + tirS strains were used in a murine model of skin infection. For preparation of the inoculum used for subcutaneous inoculation, the bacteria were grown into BHI medium at 37˚C for 8 h with constant shaking (200 rpm). They were washed twice and resuspended in sterile PBS, aliquoted to a final concentration of 1.10 7 bacteria/ml, and stored at -80˚C until used. For determination of bacterial titers, samples were serially diluted, plated on agar, and incubated overnight at 37˚C. Viability of the inocula was confirmed by colony counts with each experiment. Moreover, to ensure that the results of experiments were consistent, hemolysis phenotype was checked using blood agar plates (Trypcase soy agar + 5% sheep blood, bioMe ´rieux, France).

Mouse strains. Mice on a C57BL/6 genetic background were used in all experiments. Sixweek-old WT female mice were purchased from Charles River, France. Male and female MyD88-deficient mice were kindly provided by L. Genestier (from S. Akira's lab; [56]). All mice were maintained in pathogen-free conditions in a biosafety level 2 facility at the Plateau de Biologie Expe ´rimentale de la Souris (PBES, Ecole Normale Supe ´rieure de Lyon, Lyon, France).

Skin lesion model. One day prior to infection, mice were prepared for inoculation. Animals first were anesthetized with 2% isoflurane, and a flank was shaved with electric clipper and hair remover cream (Nair, Church & Dwight Co. Inc., Princeton, NJ) on the shaved flank. On the day of infection, mice were infected subcutaneously with 100 μl of the bacterial suspension (1.10 6 CFUs) in the shaved area. This inoculum was determined in preliminary studies to produce consistent skin lesions. Mice were returned to their cages and observed to awaken. All mice had free access to food and water throughout the duration of the experiments. Animals were weighed at 24 h intervals for 14 days. The area of lesions was measured daily using an electronic caliper and calculated with the formula A = π × (L/2) × (W/2) (mm 2 ). PBS injection was used for controls.

Bacterial recovery and cytokine quantification in skin lesions. To determine the number of CFUs at the site of infection, a second set of mice was inoculated as described above. On days 5 to 9 after infection, mice were euthanized by cervical dislocation; the lesion and the surrounding tissues were removed and transferred in sterile tubes. Tissue samples were weighed, homogenized in 1 ml of PBS (gentleMACS Dissociator, Miltenyi Biotec, Germany), diluted in sterile PBS, and plated on selective agar (ChromID S. aureus, bioMe ´rieux, France).

Enumeration of CFUs was performed 24 h later. For determination of cytokine and MPO levels, lesion homogenates were centrifuged and the supernatant removed and immediately stored at -80˚C. Cytokine levels were determined using Luminex assays (Bio-Techne) according to the manufacturer's instructions. The amount of MPO in the skin lesions was quantified using a commercially available ELISA kit (Bio-Techne).

Statistical analysis

The data were analyzed using R software (http://www.r-project.org). Student's t test, Wilcoxon test, or one-way ANOVA followed by multiple comparisons tests (Tukey) were used to compare tirS expression, luciferase activity, lesion size, cytokine levels, and bacterial CFUs between the groups. Correlations were assessed by Spearman's correlation. In all experiments, values of p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.

Abstract

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/ interleukin 1 receptor (TIR) domain-containing protein (PumA) of the multi-drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF-jB, a property transferable to non-PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll-like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin-associated protein 1 (UBAP1), a component of the endosomal-sorting complex required for transport I (ESCRT-I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.

Introduction

Microbial pathogen recognition by innate immune receptors initiates a progression of molecular interactions and signalling events assuring host defence. In bacterial infections, detection of surface components, such as peptidoglycan, lipopolysaccharides and flagellin by Toll-like receptors (TLR) 2, 4 and 5, respectively, is essential for induction of pro-inflammatory cytokines and type I interferon (IFN) responses. Specific sorting and signalling adaptor proteins bridge activated receptors with downstream kinases to initiate signalling cascades via Toll/interleukin 1 receptor (TIR) domains present on both the adaptors and the cytosolic face of TLRs [START_REF] Brubaker | Innate immune pattern recognition: a cell biological perspective[END_REF]. Upon TLR2 or TLR4 activation, the TIRcontaining adaptor protein (TIRAP) recruits myeloid differentiation primary response 88 (MyD88) that interacts with the TLR via its TIR domain [START_REF] Fitzgerald | Mal (MyD88-adapter-like) is required for tolllike receptor-4 signal transduction[END_REF][START_REF] Horng | TIRAP: an adapter molecule in the Toll signaling pathway[END_REF][START_REF] Kagan | Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling[END_REF]. MyD88 oligomerization and recruitment of specific kinases leads to the formation of myddosomes, signalling platforms that induce NF-jB translocation into the nucleus and subsequent transcription of pro-inflammatory associated genes [START_REF] Nagpal | Natural loss-of-function mutation of myeloid differentiation protein 88 disrupts its ability to form myddosomes[END_REF][START_REF] Bonham | A promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction[END_REF]. TLR4 activation also results in induction of a type I IFN via another set of adaptors, TRAM and TRIF [START_REF] Fitzgerald | Mal (MyD88-adapter-like) is required for tolllike receptor-4 signal transduction[END_REF]Yamamoto et al, 2002;[START_REF] Kagan | TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta[END_REF]. In the case of the MyD88-dependent TLR5, the identity of a sorting adaptor remains undefined and the role of TIRAP unclear although it has been implicated in proper TLR5 signalling in epithelial cells [START_REF] Choi | PTEN regulates TLR5-induced intestinal inflammation by controlling Mal/TIRAP recruitment[END_REF]. Microbial pathogens have been shown to counter these host defence pathways. Most bacterial immune-modulatory proteins described to date rely on inhibition of downstream signalling components, such as MAP kinases and transcription factors (reviewed in Rosadini & Kagan, 2015). In contrast, few examples of direct blocking at the level of initial receptor-adaptor complexes are known. Some bacterial pathogens rely on TIR domain-containing proteins to perturb TIR-dependent interactions (Newman et al, 2006;Cirl et al, 2008;Salcedo et al, 2008Salcedo et al, , 2013)), essential in innate immune signalling. The growing number of bacterial TIR proteins recently identified in both Gram-negative and Gram-positive human pathogens (Spear et al, 2012;Askarian et al, 2014;[START_REF] Zou | A TIR domain protein from E. faecalis attenuates MyD88-Mediated signaling and NF-jB activation[END_REF] highlights the importance of this immune evasion strategy in disease. However, the molecular mechanisms underlying most TIRdependent virulence strategies remain to be defined.

We focused on a previously uncharacterized TIR domaincontaining protein of the multi-drug resistant pathogen Pseudomonas aeruginosa PA7, that we called PumA. P. aeruginosa PA7 lacks genes encoding the type III secretion system (T3SS) and its cognate effector proteins that are normally associated with strong induction of cell death, a hallmark of acute P. aeruginosa infections (reviewed by [START_REF] Filloux | Protein secretion systems in Pseudomonas aeruginosa: an essay on diversity, evolution, and function[END_REF]. In addition, PA7 does not show high lytic capacity towards epithelial cells due to exolysin A (ExlA) as described for the haemorrhagic pneumonia-causing strain of the same family [START_REF] Elsen | A type III secretion negative clinical strain of Pseudomonas aeruginosa employs a two-partner secreted exolysin to induce hemorrhagic pneumonia[END_REF][START_REF] Reboud | Phenotype and toxicity of the recently discovered exlA-positive Pseudomonas aeruginosa strains collected worldwide[END_REF]. We thus took advantage of the absence of traditional virulence factors in this P. aeruginosa strain to study the molecular interactions involved in TIR-mediated bacterial targeting of events proximal to receptoradaptor signalling complexes and to dissect PumA function. We found that the PumA Pseudomonas TIR domain-containing protein is essential for PA7 virulence conferring a previously unrecognized ability to Pseudomonas to down-modulate innate immune responses during infection. We show that PumA directly interacts with both TIRAP and MyD88 to control TLR signalling. Uniquely, it also targets the ubiquitin-associated protein 1 (UBAP1), a recently discovered component of the endosomal-sorting complex required for transport I (ESCRT-I; [START_REF] Stefani | UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting[END_REF]. UBAP1 is known to play a key role in selective sorting of ubiquitinated endosomal cargo on multi-vesicular bodies (MVB), via its interaction with VPS37A and other components of ESCRT-I namely TSG101 [START_REF] Wunderley | The molecular basis for selective assembly of the UBAP1-containing endosome-specific ESCRT-I complex[END_REF], as well as with the ESCRT regulator, His domain protein tyrosine phosphatase (HDPTP; [START_REF] Stefani | UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting[END_REF]. UBAP1 has been shown to control endosomal sorting of ubiquitinated EGFR [START_REF] Stefani | UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting[END_REF] as well as ubiquitin-dependent degradation of antiviral surface proteins [START_REF] Agromayor | HA-MyD88 Myc-PumA HA-MyD88 Myc-PumA Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 References[END_REF] and integrins [START_REF] Kharitidi | Interplay of endosomal pH and ligand occupancy in integrin a5b1 ubiquitination, endocytic sorting, and cell migration[END_REF]. More recently, UBAP1 was shown to modulate steady-state trafficking of cytokine receptors in nonstimulated cells [START_REF] Mamin ´ska | ESCRT proteins restrict constitutive NF-jB signaling by trafficking cytokine receptors[END_REF]. UBAP1 is expressed in a wide range of tissues, but when deleted in mice, it is lethal for embryos [START_REF] Agromayor | HA-MyD88 Myc-PumA HA-MyD88 Myc-PumA Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 References[END_REF].

We propose that this novel Pseudomonas effector modulates UBAP1 function, hence the name PumA (for Pseudomonas UBAP1 modulator A), which confers to this TIR domain-containing protein the distinctive ability to also interfere with cytokine receptor signalling. Targeting of both TLR adaptors and UBAP1 by PumA is not spatially restricted as we found UBAP1 can associate with MyD88 in host cells. Our results thus highlight a novel role of bacterial TIR domains and place UBAP1 sorting in the context of TLR signalling.

Results

PumA is required for Pseudomonas aeruginosa PA7 virulence in vivo

In Pseudomonas, TIR domain-containing proteins were first identified in an in silico study in P. aeruginosa and the plant pathogen P. syringae (Zhang et al, 2011). Analysis of currently available genomes shows that several plant strains encode such proteins as well as additional human pathogenic strains of P. stutzeri and P. aeruginosa. The closest orthologue is found in the plant pathogen P. viridiflava. The TIR domain of PumA spans the first 136 amino acids of PumA (Appendix Fig S1A and B), with no significant sequence/structure homologies detected for the C-terminal domain (amino acid 137-303) and no signal peptide. Analysis of the PA7 genome shows pumA (PSPA7_2375) is within the genomic island RGP56, which displays a G+C content of 58.5% in contrast to the average 66.5% in the remaining genome. Interestingly, using Geneious [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF], we found the pumA gene itself has an even larger reduction in G+C content (46.6%) (Appendix Fig S1C), suggesting that it is not a conserved gene within its immediate genetic context.

We assessed the potential role of PumA in virulence by infecting the nematode Caenorhabditis elegans, a well-established model for P. aeruginosa allowing for rapid assessment of virulence [START_REF] Garvis | Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis Gene cheB2 in Pseudomonas aeruginosa virulence[END_REF]. Infection with the highly virulent strain P. aeruginosa PA14 which contains virulence factors such as the T3SS but no TIR protein resulted in 50% lethality at day 5. The PA7 wild-type strain caused 50% lethality 7 days after inoculation. In contrast, we found that the PA7 ∆pumA mutant showed a slight but significant attenuation in virulence in C. elegans (Fig 1A). These differences were not due to an in vitro growth defect of the mutant (Appendix Fig S2A) nor to a problem in expression of PumA in the wild-type P. aeruginosa PA7 strain (Appendix Fig S2B).

We then used an acute in vivo infection model to evaluate the involvement of pumA in P. aeruginosa induced lung injury. Mice infected with DpumA showed a clear increased survival compared to wild-type strain (Fig 1B). A dose of 4.10 7 CFU of PA7 induced 100% lethality after 52 h against 62.5% survival after 96 h for the DpumA mutant. Bacterial clearance and cellular recruitment were then analysed with a lower inoculum of 3.10 7 CFU. PA7DpumA infected mice showed decreased cell recruitment (Fig 1C) and an enhanced lung bacterial clearance in bronchoalveolar lavages (BAL) compared to the wild-type strain (Fig 1D). The bacterial dissemination measured with the spleen bacterial load was equivalent between the two groups (Fig 1E). Together these results show that PumA is required for P. aeruginosa PA7 infection.

PumA inhibits NF-jB translocation into the nucleus during infection in vitro

As bacterial TIR proteins down-modulate NF-jB activation (Newman et al, 2006;Cirl et al, 2008;Salcedo et al, 2008Salcedo et al, , 2013;;Spear et al, 2012;Askarian et al, 2014;[START_REF] Zou | A TIR domain protein from E. faecalis attenuates MyD88-Mediated signaling and NF-jB activation[END_REF], we infected the lung carcinoma epithelial cell line A549, a wellestablished cellular model for Pseudomonas infection and analysed NF-jB translocation into the nucleus after one hour of infection by confocal microscopy. We developed an automated analysis of p65/RelA fluorescence in relation to DAPI labelling using a specific ImageJ plugin from images obtained by confocal microscopy (Fig EV1A) which allowed us to clearly differentiate between TNFa-treated and mock-infected cells (Figs 2A and EV1B). Infection with the three heat-killed P. aeruginosa strains, wild-type PA7, isogenic mutant DpumA or wild-type PA14 resulted in significant induction of NF-jB translocation into the nucleus, although to a lower level than TNFa-treated cells (Fig 2A). When cells were infected with PA7, there was no significant induction of We then investigated whether expression of PumA alone could confer the ability to block NF-jB translocation to a different Pseudomonas strain. We chose PA14, which does not contain pumA and is known to be more virulent due to the presence of several virulence factors, namely those secreted by the T3SS. As expected, cells infected with wild-type PA14 showed high levels of NF-jB translocation into the nucleus (Fig 2B). Induction of pumA from the PA14 chromosome, which did not impact membrane permeability (Fig EV2A ), was sufficient to enable this highly virulent strain to block NF-jB accumulation in the nucleus of infected cells (Fig 2B). These data indicate that PumA expression in P. aeruginosa is necessary and sufficient for NF-jB inhibition, highlighting its central role in immune evasion.

PumA translocation into host cells during infection in vitro

We next sought to determine whether PumA could be secreted by Pseudomonas. Fractionation of bacterial cells grown in liquid culture indicates that PumA is mostly cytoplasmic and to a lesser extent associated with the inner membrane (Appendix Fig S3A). The protein was not detected in the outer membrane fractions nor could it be found in the supernatant indicating absence of secretion into the extracellular milieu in vitro. To determine whether PumA could be found inside host cells during infection, we fused chromosomal PumA with the TEM1 b-lactamase. Although the presence of other b-lactamases in PA7 and/or potential bacterial lysis resulted in non-specific cleavage of the CCF2 substrate within host cells infected with the wild-type strain, significant levels of coumarin fluorescing cells following infection with a strain containing PumA-TEM1 (Appendix Fig S3B ) suggest that PumA is translocated into host cells during infection.

PumA is associated with both TIRAP and MyD88 at the plasma membrane and intracellular compartments

To determine the mechanism by which PumA interferes with NF-jB activity, PumA was expressed in mammalian cells. We found PumA localized mostly at the plasma membrane, with some intracellular distribution, independently of the tag and in both immortal HeLa cells (Fig EV3A, top panel) and primary mouse embryonic fibroblasts (MEFs,Fig EV3B). As this localization was reminiscent of that of the TLR adaptor TIRAP (Fig EV3A andB), we co-transfected cells with PumA and TIRAP. We found extensive co-localization, in particular at the plasma membrane in both HeLa and MEFs (Fig 3A and B). We observed these results with any combination of tags (HA, Myc or GFP) for both proteins.

In contrast with TIRAP, MyD88 is mostly localized in intracellular structures that do not label the plasma membrane. We therefore co-expressed MyD88 with PumA. Surprisingly, we found enrichment of PumA in a proportion of MyD88-positive structures in both cell types although to a lesser extent than that observed with TIRAP (Fig 3A andB). These results were confirmed by structured illumination microscopy (SIM) to enable imaging at higher resolution (Fig 3C andD The TIR domain of PumA is responsible for interaction with both TIRAP and MyD88

We then investigated whether the TIR domain present in the first 136 amino acids of PumA was responsible for membrane targeting. PumA1-136 was also efficiently targeted to the plasma membrane (Fig 4A). However, unlike TIRAP and another bacterial TIR protein BtpA/TcpB which are known to interact with specific phospholipids of the plasma membrane [START_REF] Kagan | Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling[END_REF]Radhakrishnan et al, 2009), PumA and PumA1-136 did not show any lipid binding properties when incubated with phosphoinositide phosphate strips (Fig 4B). We then tested whether PumA could interact with TIRAP, which could explain its membrane localization. We found that TIRAP-GFP and Myc-PumA co-immunoprecipitated (co-IP) suggesting TIRAP and PumA could be part of the same complex (Fig 4C). This association was confirmed using purified His-tagged PumA or PumA1-136 immobilized on Ni-NTA resin, which both retained HA-TIRAP (Fig 4D).

As we had also observed enrichment of PumA in MyD88-positive compartments, we investigated whether PumA could interact with this adaptor protein. Although we did not observe and interaction between GFP-MyD88 and Myc-PumA ( of PumA, which mediates interaction with these adaptor proteins via its TIR domain.

PumA interacts with the ESCRT-I component UBAP1

As PumA was able to interact with both TIRAP and MyD88, two key adaptors in TLR signalling, we hypothesized PumA's function was to block all immune pathways dependent on these adaptors. Using an in vitro luciferase assay, we tested key immune receptors implicated in Pseudomonas infection. Surprisingly, we found PumA could not only block TLR4, TLR5 and IL-1b but also the TNF receptor,

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The While we were conducting this work, another study reported UBAP1 participates in control of TNFR1 and other cytokine receptor trafficking [START_REF] Mamin ´ska | ESCRT proteins restrict constitutive NF-jB signaling by trafficking cytokine receptors[END_REF]. Our data along with this recently published study thus suggest that PumA interaction with UBAP1 results in inhibition of the TNF receptor-mediated pathway. To determine whether PumA was targeting two different types of cellular compartments, one with UBAP1 controlling the TNFR pathway and another containing TLR adaptors, we analysed whether UBAP1 was excluded from TIRAP and MyD88 containing compartments. We first analysed their intracellular localization following transfection as we were not able to detect endogenous UBAP1 with currently available antibodies. Extensive co-localization was observed at the plasma membrane and intracellular structures when co-expressing UBAP1 and TIRAP (Fig 7A ), with no visible impact on the normal distribution of TIRAP. However, in the case of MyD88, co-expression with UBAP1 resulted in accumulation of this adaptor at the plasma membrane, not seen in cells expressing MyD88 alone (Fig 7A,bottom panel and B). Quantification of membrane enrichment of MyD88 in cells expressing UBAP1 showed MyD88 membrane association was even more striking in the presence of UBAP1 (Fig 7C ) than that observed when co-expressing TIRAP with MyD88 [START_REF] Kagan | Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling[END_REF], suggesting UBAP1 could be participating in MyD88 intracellular sorting.

To determine whether UBAP1 could be interacting with these TLR adaptors, we carried out biochemical analysis of cells coexpressing UBAP1 and either TIRAP or MyD88. We could efficiently detect an interaction between UBAP1 and MyD88 by co-IP (Appendix Fig S5D andE) but not UBAP1 and membrin (Appendix Fig S5E), used as a control eukaryotic protein with the same tag. In the case of TIRAP, the co-IP was much less efficient (Appendix Fig S5D). These results suggest that UBAP1 may be associated with MyD88-containing compartments and to a lesser extent TIRAP, consistent with our microscopy observations. To confirm these results and ensure these interactions were taking place with UBAP1 in the context of the ESCRT-I, we determined whether MyD88 and TIRAP could interact with endogenous UBAP1 and TSG101. We found that HA-MyD88 co-immunoprecipitated both components of the ESCRT-I as well as endogenous TIRAP (Fig 7D ), as expected. However, we did not observe an interaction between HA-TIRAP and endogenous UBAP1 nor TSG101 (Fig 7E ), suggesting that only MyD88 can be found associated with the ESCRT-I.

Overall, these data suggest that PumA mediates interactions with UBAP1 in the context of ESCRT-I, which can itself associate with the TLR adaptor MyD88, also targeted by this P. aeruginosa effector protein.

Pseudomonas aeruginosa PA7 induces a decrease of TNFR1 in a PumA-dependent manner during infection in vitro It is well described in the literature that inhibition of UBAP1 induces intracellular accumulation of EGFR, LTbR and TNFR1 [START_REF] Stefani | UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting[END_REF][START_REF] Mamin ´ska | ESCRT proteins restrict constitutive NF-jB signaling by trafficking cytokine receptors[END_REF]. To establish a link between PumA interaction with UBAP1 and the ability of PumA to reduce TNFadependent signalling (Fig EV5A ), we analysed the levels of TNFR1 during infection. In wild-type PA7 infected A549 cells, we observed a decrease of TNFR1 compared to the negative control (Fig 7F). In contrast, the mutant lacking PumA was not able to reduce the levels of TNFR1 in infected cells and this phenotype could be fully restored by expression of PumA in the complemented strain. This is consistent with PumA targeting of UBAP1 and enhancing its activity during infection in vitro. Interestingly, we did not see any impact on the overall levels of TIRAP during infection (Fig 7F ) suggesting that PumA is not inducing TIRAP degradation as was previously reported for BtpA (Sengupta et al, 2010).

Discussion

Many pathogens have developed sophisticated strategies to evade or modify host immune responses to their advantage. We have found that the TIR domain-containing protein PumA plays a major role in the virulence of multi-drug resistant P. aeruginosa PA7 strain. PumA ensures efficient inhibition of innate immune responses by interacting with MyD88 and TIRAP, key adaptor proteins for IL-1R and the main relevant TLRs in Pseudomonas infection (TLR4 and TLR5), as well as UBAP1 which regulates cytokine receptor pathways. These results identify UBAP1 as a novel cellular target for bacterial pathogens.

Pseudomonas aeruginosa is an important human pathogen associated with high level of mortality in nosocomial infections and cystic fibrosis patients. Most P. aeruginosa strains rely on a multitude of virulence factors to control host cellular pathways, including effectors delivered by the T3SS. However, in a cystic fibrosis context, colonizing strains modulate levels of expression of some of these virulence factors [START_REF] Hauser | Clinical significance of microbial infection and adaptation in cystic fibrosis[END_REF], namely downregulation of the T3SS [START_REF] Jain | Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis[END_REF] and undergo a remarkable accumulation of pathoadaptive mutations [START_REF] Marvig | Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis[END_REF]. The Myc-UBAP1 HA-MyD88 E PA7-related P. aeruginosa strains lack the 20-Kb-long genomic region encoding the T3SS core components and all genes encoding secreted effectors but contains several additional genomic islands and potential novel virulence factors [START_REF] Pirnay | Pseudomonas aeruginosa population structure revisited[END_REF][START_REF] Roy | Complete genome sequence of the multiresistant taxonomic outlier Pseudomonas aeruginosa PA7[END_REF][START_REF] Cadoret | Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/ TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein[END_REF][START_REF] Freschi | Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium[END_REF]. In some of these strains, such as CLJ1, an exolysin secreted by a two-partner secretion system is responsible for hypervirulence [START_REF] Elsen | A type III secretion negative clinical strain of Pseudomonas aeruginosa employs a two-partner secreted exolysin to induce hemorrhagic pneumonia[END_REF]. However, in PA7, this exolysin is detected at only low levels in the secretome and is not responsible for cytotoxicity [START_REF] Reboud | Phenotype and toxicity of the recently discovered exlA-positive Pseudomonas aeruginosa strains collected worldwide[END_REF], suggesting an alternative pathogenicity mechanism. In this context, we hypothesize that PumA might be underlying an alternative pathogenicity mechanism to allow PA7 persistence within a host. Consistently, we observed a clear attenuation in virulence for a PA7 strain lacking pumA in both C. elegans and in a mouse lung infection model. Interestingly, no impact in the ability of the pumA mutant to disseminate systemically was observed suggesting a role in control of local pathology. This type of P. aeruginosa infection based on persistence and colonization rather than rapid cytotoxicity could be relevant in specific clinical contexts such as infection of wound and burn patients, aggravated by the high level of multi-drug resistance. It is interesting to note that other Pseudomonas contain a TIR domain protein, namely several strains pathogenic in plants. In this context, it will be interesting to analyse the role of the orthologous TIR protein in the plant pathogens P. syringae or P. viridiflava with over 90% identity to PumA in amino acid sequence for the TIR domain, regarding control of plant responses as these functions may be relevant across taxonomic kingdoms.

Pseudomonas is not the only bacterial pathogen to take advantage of the TIR domain to engage TIR-TIR interactions which are essential components of innate immune signalling. Bacterial targeting of TLRs has been best described for uropathogenic E. coli TcpC (Cirl et al, 2008) and Brucella BtpA, also known as TcpB (Cirl et al, 2008;Salcedo et al, 2008), even though their molecular mode of action remains elusive. Brucella relies on an additional TIR protein, BtpB to down-modulate inflammation during infection (Salcedo et al, 2013). TcpC was shown to interfere with MyD88-dependent and independent pathways to down-modulate TLR signalling and contribute to kidney pathology (Cirl et al, 2008;Yadav et al, 2010). In the case of Brucella, BtpA/TcpB has been described as a mimic of TIRAP, since it can directly bind specific phosphoinositides of the plasma membrane (Radhakrishnan et al, 2009). This is clearly distinct from PumA that shows no significant lipid binding properties. In addition, BtpA/TcpB was also shown to bind TIRAP, which results in its increased ubiquitination and degradation during infection (Sengupta et al, 2010), which also differs from PumA which despite TIRAP binding does not induce its degradation. Several studies have followed disputing the precise target of BtpA/ TcpB with some proposing preferential binding to MyD88 (Chaudhary et al, 2011). One key question that remains unanswered is how these bacterial TIR proteins are entering host cells and where do they localize during infection? No direct imaging of bacterial TIR proteins has been described. In the case of TcpC, internalization into host cells was observed but the export mechanism was not identified (Cirl et al, 2008), whereas no data are available regarding Salmonella, Yersinia, Staphyloccocus and Enteroccoccus. In the case of Brucella, depending on the fusion tags, translocation into host cells of BtpA/TcpB or BtpB was dependent or independent of the T4SS (Salcedo et al, 2013) whereas a separate group has proposed that BtpA/TcpB is cell permeable and may enter host cells in a passive manner [START_REF] Radhakrishnan | Biochemical and functional analysis of TIR domain containing protein from Brucella melitensis[END_REF]. Unfortunately, PumA fusion with CyaA resulted in its cleavage preventing us from using this system. Using different fluorescent tags and the specific anti-PumA antibody, we were not able to confidently visualize it inside host cells during infection. We were however able to detect intracellular PumA using a TEM1 fusion. Further work needs to be carried out to confirm translocation of PumA into host cells and define the intracellular location of PumA during infection. PumA was also not found in the bacterial culture supernatant in vitro, suggesting that contact-dependent delivery is involved. How PumA is entering host cells will have to be further investigated, but since the T3SS is not present in PumA-encoding strains, it suggests that host cell delivery would need an alternative secretion pathway.

It is important to note that TIR domains are widespread in multicellular organisms, such as in plants (role in disease resistance) and amoebas (dual role in ingestion of bacteria and immune-like functions) as well as in numerous bacterial genera that include cyanobacteria and other non-pathogenic bacteria (Zhang et al, 2011). This suggests these domains have evolved as an essential protein-protein interaction platform that could have additional functions. Indeed, recently the TIR domain of TcpC has been shown to directly interact with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome and caspase-1, besides MyD88, to perturb inflammasome activation (Waldhuber et al, 2016). There are also additional potential targets yet to be identified for BtpA/TcpB since it interferes with microtubule dynamics (Radhakrishnan et al, 2011;Felix et al, 2014) and induces unfolded protein response (Smith et al, 2013). Western blot of TNFR1 in A549 cells infected for 1 h with either P. aeruginosa PA7 wt, ∆pumA or ∆pumA:pumA (Ara) induced with 1% arabinose. A mock-infected sample was included as a negative control. The same blot was also probed for TIRAP and actin to control loading.

Source data are available online for this figure.

This notion that bacterial TIR domains provide a broad interaction platform is supported by our observations. We found that in addition to directly interacting with TIRAP and MyD88, PumA also targets the ESCRT-I machinery by binding to UBAP1 as PumA could co-immunoprecipitate endogenous UBAP1 and TSG101. All these interactions seem to be mediated by the TIR domain of PumA, but endogenous co-IP experiments showed that the full-length PumA is required for efficient interactions to occur. It is likely that TIR-TIR interactions are taking place with TIRAP and MyD88. In the case of UBAP1, the PumA interacting domain remains to be identified. All yeast two-hybrid preys identified in our screen encoded for a region containing amino acid 45-164, present between two key functional domains: the N-terminal UBAP1-MVB12-associated (UMA) domain (residues 17-63) that binds the central stalk of ESCRT-I Vps37 and the central domain (residues 159-308), containing the recently identified key binding site for HDPTP which can act as a cargo adaptor [START_REF] Gahloth | Structural basis for selective interaction between the ESCRT regulator HD-PTP and UBAP1[END_REF]. The C-terminal portion of UBAP1 includes a SOUBA domain (residues 381-502) known to bind ubiquitin [START_REF] Agromayor | HA-MyD88 Myc-PumA HA-MyD88 Myc-PumA Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 References[END_REF]. UBAP1 is a key component of ESCRT-I that enables sorting of ubiquitinated cargo on MVBs. PumA may be binding an intermediate region of UBAP1 that could partially overlap with that interacting with HDPTP. Further work is now necessary to confirm this hypothesis. In view of the recent work implicating UBAP1 in restriction of constitutive NF-jB signalling (Mamin ´ska et al, 2016), PumA could be impacting the activation of TNFR pathway through UBAP1. Depletion of UBAP1 was shown to induce intracellular accumulation of the cytokine receptors in endosomal compartments [START_REF] Mamin ´ska | ESCRT proteins restrict constitutive NF-jB signaling by trafficking cytokine receptors[END_REF], which leads to increase in constitutive levels of NF-jB, since UBAP1 cannot ensure proper steady-state cytokine receptor (such as LTbR and TNFR1) sorting and subsequent degradation. Since in vitro experiments suggest PumA is blocking TNF receptor-mediated pathway, PumA could be enhancing activity of UBAP1. This phenotype is specific of PumA since we observed no effect of another bacterial TIR domain-containing protein BtpA/TcpB which does not interact with UBAP1 and its ectopic expression does not result in inhibition of the TNF-induced pathway. Consistent with our hypothesis, wild-type PA7 decreases the levels of TNFR1 in A549 cells in a PumAdependent manner suggesting targeting of UBAP1 is occur-ring during infection and could enhance its activity.

In an attempt to determine whether distinct intracellular locations were targeted by PumA to enable interaction with TLR adaptors and the ESCRT-I component UBAP1, we analysed whether UBAP1 was excluded from TIRAP or MyD88-enriched compartments. Surprisingly, co-IP experiments revealed endogenous UBAP1 itself and TSG101 could be found associated with MyD88 but not TIRAP, suggesting that the ESCRT-I machinery may be interacting with specific TLR adaptors. We therefore propose that additional crosstalk between these pathways may exist. MyD88 has been shown to interact with TLRs and with TIRAP via its TIR domain or the death domain. It remains to be demonstrated whether UBAP1 interacts directly with MyD88 but our data strongly suggest they can be found in the same complex, namely at the plasma membrane. Interestingly, co-expression of MyD88 and UBAP1 resulted in MyD88 enhanced plasma membrane targeting, to higher levels than that previously described for TIRAP [START_REF] Kagan | Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling[END_REF]. Further work is required to determine if UBAP1 interaction with MyD88 promotes activation of TLR signalling and whether PumA could disrupt this interaction. A few studies have suggested the implication of ESCRT-I or MVBs in the control of TLR pathways. In Drosophila, ESCRT-0 components modulate endosomal sorting of Toll [START_REF] Husebye | Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity[END_REF][START_REF] Huang | Endocytic pathway is required for Drosophila Toll innate immune signaling[END_REF]. ESCRT have been also shown to negatively regulate TLR7 and 9 to enable recycling of these receptors following ubiquitination [START_REF] Chiang | Cofactors required for TLR7-and TLR9-dependent innate immune responses[END_REF]. More interestingly, inhibition of endosomal sorting via ESCRT-I increases LPS-induced signalling [START_REF] Husebye | Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity[END_REF], suggesting it is playing a role in sorting and degradation of activated receptor complexes.

In conclusion, our study describes a P. aeruginosa effector PumA that targets UBAP1 in the context of ESCRT-I and plays a major role in virulence. In addition, our data associate UBAP1 to MyD88, highlighting a potential larger role of endosomal sorting by ESCRT-I in regulation of TLR signalling.

Materials and Methods

Strains

Pseudomonas aeruginosa strains used in this study were wild-type PA7, PA14 or derived strains and were routinely cultured in liquid Luria Bertani (LB) medium. Antibiotics were added to P. aeruginosa cultures, when appropriate, at the following concentrations: 150 lg/ ml tetracycline and 750 lg/ml carbenicillin. When indicated, arabinose at 1% or glucose at 0.5% was added to cultures. For Escherichia coli cultures, antibiotics were added when necessary at the following concentrations: 50 lg/ml kanamycin and 50 lg/ml ampicillin.

Construction of Pseudomonas DpumA mutant and complemented strains

The 500 base pairs upstream and 500 base pairs downstream of pumA gene (PSPA7_2375; NC_009656.1.) were amplified from P. aeruginosa PA7 genomic DNA to do overlapping PCR, using primers 5 0 -TTTGGGCCCAAGACGATCAGCGGCACC-3 0 , 5 0 -ATCGGCT CTGCCCTATGCCATCTTTTTAACTCCATCCTTGTAATTCC-3 0 , 5 0 -GG ATGGAGTTAAAAAGATGGCATAGGGCAGAGCCGAT-3 0 and 5 0 -TT TTGATCACAACTACCCCGATGCGTT-3 0 , respectively. Then, the PCR product was sub-cloned into pGEM ® -T Easy Vector (PROMEGA) and ligated into pKNG208 [START_REF] Cadoret | Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/ TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein[END_REF] following digestion with SpeI and ApaI to generate pKNG208-∆pumA. This plasmid was introduced into P. aeruginosa PA7 by conjugation where it is incapable of autonomous replication. Homologous recombination events were primary selected using tetracyclin resistance (150 lg/ml) in Pseudomonas isolation agar (PIA) plates and secondary selected using sucrose 6% sensitivity in LB agar plates during 2-3 days at room temperate. PCR and sequencing analyses confirmed the pumA wild-type gene was deleted and Western blotting showed absence of PumA production of the PA7 ∆pumA strain (Appendix Fig S2B).

The mini-CTX-PBAD plasmid was constructed by cloning the SalI-AraC-PBAD-SacI fragment from pJN105 vector [START_REF] Newman | Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator[END_REF] into the 6711 bp SalI/SacI DNA fragment from miniCTX-lacZ vector [START_REF] Hoang | A broadhost-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants[END_REF]. PSPA7_2375 gene was amplified with an artificial Shine-Dalgarno (AAGAAG) and cloned into mini-CTX-PBAD digested by SpeI/SacI using the SLIC method [START_REF] Jeong | Onestep sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies[END_REF].

Primers used were 5 0 -AGCCCGGGGGATCCACTAGTAGGAGGTGA GATATACAATGGCGGTCTTCATTAGTTA-3 0 and 5 0 -ACCATCCAGT GCAGGAGCTCCTATGCGCGCGGCCACGGG-3 0 .

Construction of PA7 pumA::bla1 strain

The 500 base pairs upstream and downstream of pumA stop codon from P. aeruginosa PA7 genomic DNA and blaM gene from pJC121 plasmid (Myeni et al, 2013) were PCR amplified using primers 5 0 -ATTACGCGTTAACCCGGGCCCAGGATGTTGACGGCTATC-3 0 , 5 0 -CAGCGTTTCTGGTGCGCGCGGCCACGG-3 0 , 5 0 -CTGATTAAGTAGGG CAGAGCCGATCAGCTC-3 0 , 5 0 -ACACTGGCGGCCGTTACTAGTGCTG GACTGGCGCAACTA-3 0 , 5 0 -TGGCCGCGCGCACCAGAAACGCTGGT GAAA-3 0 and 5 0 -ATCGGCTCTGCCCTACTTAATCAGTGAGGCACC T-3 0 and used in overlapping PCR. DNA product was then cloned by the SLIC method [START_REF] Jeong | Onestep sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies[END_REF] into pKNG208 [START_REF] Cadoret | Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/ TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein[END_REF] digested by ApaI/SpeI to generate pKNG208-pumA::bla1 vector.

Construction of eukaryotic expression vectors

The PumA constructs were obtained by cloning in the gateway pDONR TM (Life Technologies) and then cloned in the pENTRY Myc, HA or GFP vectors. The following primers were used 5 0 -GGGGA CAAGTTTGTACAAAAAAGCAGGCTTCGCGGTCTTCATTAGTTATT CCCACG-3 0 and 5 0 -GGGGACCACTTTGTACAAGAAAGCTGGGTCC TATGCGCGCGGCCACGGGGTAGC-3 0 . PumA1-136 was constructed with the following primers: 5 0 -GGGG ACAAGTTTGTACAAAAAA GCAGGCTTCATGGCGGTCTTCATTAGTTATTCC -3 0 ; 5 0 -GGGGACCA CTTTGTACAAGAAAGCTGGGTCCTAACGGGACTGATCAGGATTAG AG-3 0 . PumA137-303 with 5 0 -GGGGACAAGTTTGTACAAAAAAGCA GGCTTC ATTGAGGATGTTGACGGCTA-3 0 ; 5 0 -GGGGACCACTTTG TACAAGAAAGCTGGGTC CTATGCGCGCGGCCACGGGGTAGC -3 0 .

Construction of prokaryotic expression vectors

The full-length P. aeruginosa PA7 pumA and its TIR domain (residues 1-136) were cloned into pET151/D-Topo (Invitrogen)-which carries the T7 promoter, N-terminal 6xHis and V5 tags, protease recognition site for tobacco etch virus (TEV) protease and ampicillin resistance gene. The following primers were used: 5 0 -CAC CATGGCGGTCTTCATTAGTTATTCC-3 0 and 5 0 -TGATCGGCTCT GCCCTATGC-3 0 for pumA; the same forward primer and 5 0 -CTAACGGGACTGATCAGGATTAGAG-3 0 for pumA TIR domain. BtpA was cloned in this same vector. The HA-TIRAP and HA-Myd88 vector was used as a template to clone TIRAP and Myd88, respectively, into pRSF-MBP vector. This vector corresponds to pRSFDuet-1 (Novagen) but modified to insert 6xHis-MBP from pETM-41 vector (EMBL) behind the cloning multiple site.

Cell culture and transfections

HeLa, HEK 293T and A549 cells (all obtained from ATCC) were grown in DMEM supplemented with 10% of foetal calf serum (FCS). Mouse embryonic fibroblasts were prepared as described previously [START_REF] Conner | Mouse embryo fibroblast (MEF) feeder cell preparation[END_REF] and maintained in DMEM supplemented with 10% (FCS). All cells were transiently transfected using Fugene (Roche) for 24 h, according to manufacturer's instructions.

Pseudomonas infection of A549 cells

For adhesion assays and microscopy analysis of NF-jB, cells were first seeded into 24-well tissue culture plates at 2 × 10 5 cells/well (to obtain a monolayer) or 5 × 10 4 cells/well, respectively. Cells were infected with overnight cultures at a MOI of 10 or 100 of P. aeruginosa in 500 ll of complete medium per well. Plates were centrifuged at 400 × g for 5 min and then incubated for 1 h at 37°C with 5% CO2 atmosphere. Cells were then washed five times with DMEM and either lysed or fixed. In the case of the cytotoxicity assays, cells were incubated for longer periods with complete media. When indicated, arabinose at 1% or glucose at 0.5% was added.

For NF-jB experiments, exponential phase cultures were also used, but no differences were detected. After 1 h, medium was removed and cells were washed two times with ice-cold PBS. Control samples were always performed by incubating cells with mock inocula and following the exact same procedure as for the infection.

For adhesion assays, cells were lysed with 500 ll of 0.1% Triton solution and pipetted vigorously several times. Lysed samples were harvest, and serial 10-fold dilutions in PBS were plated on LB agar to enumerate CFUs.

For Western blot analysis of TNFR1, cells were seeded in six-well plates at 2 × 10 5 cells/well and infected as described above. At 1 h post-infection, cells were washed with ice-cold PBS 2 times, were collected and lysed directly with loading buffer. For each sample, six wells were pooled.

Cell cytotoxicity exerted by bacteria was quantified with the cytotoxicity detection kit-LDH (Roche), which measures the activity of cellular lactate dehydrogenase released into the supernatants. The assays were performed according to the instructions of the manufacturer.

For propidium iodide staining, A549 cells were maintained in DMEM media supplemented with 10% FCS. Cells were seeded at 1 × 10 5 cells/ml in 96-well plate to achieve confluent monolayers. Cells were then infected with overnight cultures of P. aeruginosa or mutants supplemented with arabinose to a final concentration of 2% (as indicated) at a MOI of 100. The plates were centrifuged at 400 × g for 5 min and incubated for 1 h at 37°C. After 1 h of infection, cells were washed three times with PBS then incubated with complete media (without red phenol) containing propidium iodide and labelling measured during 6 h every 15 min with a Tecan Infinite M1000.

Immunofluorescence labelling and microscopy

Cells were fixed in Antigenfix (DiaPath), at room temperature for 10 min. Cells were then labelled at RT with primary antibody mix diluted in 0.1% saponin in PBS with 1% BSA and 10% horse serum for blocking. Primary antibody was incubated for 1 h followed by two washes in 0.1% saponin in PBS. Secondary antibodies were then mixed and incubated for a further 30 min, followed by two washes in 0.1% saponin in PBS, one wash in PBS and one wash in distilled water before mounting with Prolong Gold. Samples were examined on a Zeiss LSM710 or Zeiss LSM800 laser scanning confocal microscopes for image acquisition. Images of 1,024 × 1,024 pixels were then assembled using plugin FigureJ from ImageJ.

For immunofluorescence microscopy analysis of NF-jB, cells were permeabilized for 6 min with 0.1% Triton in PBS, followed by a blocking for 1 h with 2% BSA in PBS. Primary antibodies were incubated for 1 h followed by two washes in 2% BSA in PBS, 30min incubation for secondary antibodies, two washes in 2% BSA in PBS, one wash in PBS and one wash in water before mounting with Prolong Gold (Life Technologies). Samples were examined on a Zeiss LSM710 laser scanning confocal microscope for image acquisition. Images of 2,648 × 2,648 pixels were then passed through a specific plugin of ImageJ developed by L. Plantevin, based on a previous study [START_REF] Noursadeghi | Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages[END_REF]; raw images were treated with a median filter and threshold moments, afterwards total NF-jB was subtracted from the Dapi channel to obtain cytoplasmic NF-jB. Total NF-jB was then subtracted from cytoplasmic NF-jB to obtain nuclear . Quantification was always done by counting at least 200 cells per condition in minimum three independent experiments, for a total of at least 600 host cells analysed per condition.

Antibodies and reagents

Primary antibodies used were rabbit anti-p65 from Santa Cruz (clone C-20, ref. sc-372) TNFR1 (Santa Cruz, and rabbit anti-TSG101 (Atlas Antibodies, ref. HPA006161) both at 1/200. Rabbit polyclonal anti-PumA serum was obtained by repeated immunization of rabbits with purified PumA (Eurogentec) and was used at 1/1,000 for Western blot and for immunofluorescence microscopy. Purified BtpA was used to obtain chicken anti-BtpA (Eurogentec). Anti-EF-Tu antibody (kind gift from R. Voulhoux) was used at 1/10,000.

Secondary antibodies used were anti-rabbit, mouse, chicken or rat conjugated with Alexas-488, -555 or -647 all from Jackson ImmunoResearch. When necessary, phalloidin-568 (1/1,000) was used to label the actin cytoskeleton and DAPI nuclear dye (1/1,000) for the host cell nucleus. For Western blots, anti-mouse or rabbit-HRP antibodies were used at 1/5,000.

TEM translocation assay

HeLa cells were seeded in a 96-well plates at 1 × 10 4 cells/well overnight. Cells were then infected with an MOI of 100 by centrifugation at 4°C, 400 g for 5 min and 1 at 37°C 5% CO2. Cells were washed with HBSS containing 2.5 mM probenecid. Then, 6 ll of CFF2 mix (as described by Life Technologies protocol) and 2.5 mM probenicid were added to each well, and incubated for 1.5 h at room temperature in the dark. Cells were finally washed with PBS, fixed using Antigenfix and analysed immediately by confocal microscopy (Zeiss LSM800) or flow cytometry (MACSQuant10 analyser).

Luciferase activity assay

HEK 293T cells were seeded in a 96-well plates at 2 × 10 4 cells/well overnight, and cells were transiently transfected with FuGENE ® 6 (Promega) for 24 h for a total of 0.4 lg of DNA consisting of 50 ng TLR plasmids, 200 ng of pBIIXLuc reporter plasmid, 5 ng of control Renilla luciferase (pRL-null, Promega) and 50 ng of myc-PumA expression vector. The total amount of DNA was kept constant by adding empty vector. Where indicated, cells were treated with E. coli LPS (1 lg/ml) and Flagellin FLA-ST (1 lg/ml), all obtained from InvivoGen, for 6 h. In the case of IL-1b and TNFR, endogenous receptors were stimulated with IL-1b (100 ng/ml) and TNFa (100 ng/ml), respectively. Cells were then lysed and luciferase activity measured using Dual-Glo Luciferase Assay System (Promega).

Yeast two-hybrid screen

Full-length pumA cloned in pB27 (N-LexA-bait-C fusion) was used in a ULTImate screen against a human normal lung-RP1 library (Hybrigenics).

Protein expression and purification

Escherichia coli BL21 star (DE3) cells carrying pET151D topo-pumA, pET151D topo-pumA1-136, or pRSFDuet-TIRAP or MyD88 plasmids were grown in 1 L Luria Bertani (LB) media containing ampicillin or kanamycin according to the plasmid at 37°C until an OD600 value of 0.5-0.8 was reached. Isopropyl-b-D-thiogalactopyranoside (IPTG) was added to final concentration of 1 mM, and culture was further grown overnight at 20°C. Cells were harvested by centrifugation at 6,000×g for 20 min at 4°C. Bacterial pellets were lysed by sonication in cold lysis buffer (40 mM Tris-HCl pH8, 250 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100) supplemented with DNase-I, lysozyme and protease inhibitor tablets (Roche). Extracts were cleared at 16,000 × g for 20 min at 4°C and loaded onto a 5 ml His-Trap column or 5 ml MBP-Trap column (GE Healthcare) pre-equilibrated with buffer A (40 mM Tris [pH8], 250 mM NaCl, 5% glycerol). The column was washed successively with buffer A, 10% v/v buffer B (buffer A with 500 mM imidazole), 1M NaCl and eluted in a gradient of buffer B (His-Trap) or wash in buffer A and eluted in buffer A containing 20 mM maltose (MBP-Trap).

Proteins used for lipid binding assay were incubated with TEV protease, 1mM DTT and 0.5 mM EDTA, dialysed against buffer A at 4°C overnight. The untagged recombinant protein was purified through a second His-trap column. Pure fractions were pooled, concentrated and applied to size exclusion chromatography (Superdex 75 10/300; GE Healthcare).

Fractions were analysed by SDS-PAGE.

Pull-downs from cell extracts

Human embryonic kidney (HEK) 293T cells were seeded at 5 × 10 5 in 10-cm cell culture dish in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% foetal bovine serum. Cells were incubated overnight in a 37°C humidified atmosphere of 5% CO2. Cells were transiently transfected with different plasmids (8 lg) using FuGENE 6 (Promega). 22 h after infection, cells were washed in ice-cold PBS, harvested and resuspended in 200 ll of RIPA buffer (Sigma) supplemented with phenylmethylsulfonyl fluoride (Sigma) and protease inhibitor

The EMBO Journal UBAP1 targeting by Pseudomonas TIR domain protein

Paul RC Imbert et al cocktail (Roche). Extracts were then centrifuged at 17,000 g at 4°C for 20 min. The supernatant was incubated with 50 lg of His tag recombinant protein during 2 h at 4°C, then incubated within gravity flow column (Agilent) containing 80 ll Ni-NTA agarose beads (Macherey-Nagel) during 1 h beforehand washed in water and pre-equilibrated in equilibrium buffer 20 mM Tris-HCl pH7.5, 250 mM NaCl. The column was washed successively three times in equilibrium buffer supplemented with 25 mM imidazole, three times in equilibrium buffer and eluted in equilibrium buffer supplemented with 500 mM imidazole. Proteins eluted were separated by SDS-PAGE, transferred to a PVDF membrane, incubated with specific primary antibodies for 1 h and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using Clarity TM Western ECL Blotting Substrate (Bio-Rad).

Co-immunoprecipitations

HEK 293T cells were cultured in 100 mm × 20 mm cell culture dishes at 4 × 10 5 cells/dish overnight. Cells were transiently transfected with 14.7 ll of Torpedo DNA (Ibidi) for 24 h for a total of 5 lg of DNA/plate. On ice, after two washes with cold PBS, cells were collected by with a cell scraper and centrifuged at 80 g at 4°C during 5 min. Cell lysis and processing for co-immunoprecipitation were done as described by either GFP-Trap ® _A kit (Chromotek) or with the PierceTM HA Epitope Antibody Agarose conjugate (Thermo scientific).

For endogenous co-IP, HeLa cells were cultured in 100 mm × 20 mm cell culture dishes at 1 × 10 6 cells/dish overnight. Cells were transiently transfected and collected as described above. Cell lysis and processing for co-immunoprecipitation were done following the manufacturers' instructions (PierceTM HA Epitope Antibody Agarose conjugate, Thermo scientific) but using 100 ll of beads and increasing the number of washes to 5.

Co-expression analysis

Escherichia coli BL21 star (DE3) cells harbouring both pET151D topo-pumA1-136 and pRSF-Duet vector-TIRAP (or Myd88 or empty vector) plasmids were grown in LB media containing ampicillin and kanamycin at 37°C until an OD600 value of 0.5-0.8 and induced with 2 mM isopropyl-b-D-thiogalactopyranoside overnight at 20°C.

Cells were lysed and loaded onto a 5 ml MBP column as described in the protein expression and purification section. Fractions were analysed by SDS-PAGE.

Lipid binding assays

Lipid binding assays were performed as described previously [START_REF] Marek | Phosphoinositide binding by the Toll adaptor dMyD88 controls antibacterial responses in Drosophila[END_REF]. Briefly, phosphoinositide phosphate (PIP) strips (Echelon Biosciences) were saturated in blocking buffer (10 mM Tris [pH8], 150 mM NaCl, 0.1% Tween 20, 0.1% Ovalbumin) for 1 h at room temperate under shacking. Strips were probed for 2 h at room temperate with each recombinant protein (2.5 lg) in the presence of the specific anti-protein antibody. PIP strips were then washed in blocking buffer three times for 10 min each and probed with an HRP-conjugated anti-rabbit IgG or anti-Hen IgY for 30 min in blocking buffer. Bound protein was detected using Clarity TM Western ECL Blotting Substrate.

Caenorhabditis elegans infection

The slow killing assay was performed as described previously [START_REF] Garvis | Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis Gene cheB2 in Pseudomonas aeruginosa virulence[END_REF]. Each independent assay consisted of three replicates. Briefly, five 60 mm NGM plates were inoculated with 60 ll of overnight culture of each bacterial strain and incubated at 37°C overnight. Plates were seeded with L4 stage hermaphrodite fer-15 worms (10 per plate). Plates were then incubated at 25°C and scored each day for live worms. A worm was considered dead when it no longer responded to touch. Escherichia coli was used as a control. Animal survival was plotted using GraphPad Prism version 6.0 for Mac, GraphPad Software, La Jolla, California, USA. Survival curves are considered significantly different from the control when P-values are < 0.05. Prism calculates survival fractions using the product limit (Kaplan-Meier) method. Prism compares survival curves by two methods: the log-rank test (also called the Mantel-Cox test) and the Gehan-Breslow-Wilcoxon test.

Mouse model of Pseudomonas acute infection

Wild-type C57BL6/J male mice, 8-10 weeks old, were purchased from Janvier laboratories. Mice were randomized before the experiments and infection were performed blindly. Following a light anaesthesia with isoflurane (Baxter), a pulmonary infection model was induced by intranasal instillation with 3 × 10 7 CFU of P. aeruginosa PA7 or PA7DpumA strains (except for survival studies conducted with lethal inocula of 4 × 10 7 CFU/mouse). All mice were sacrificed at 24 h or survival was monitored for 96 h.

To establish bacterial burden, mouse lungs and spleens were homogenized in sterile tubes with PBS. Lung and spleen homogenates were sequentially diluted and cultured on Lysogeny Broth agar plates for 24 h at 37°C to assess bacterial load. Bronchoalveolar lavage (BAL) was done as follows: lungs from each experimental group were washed with a total of 1.5 ml sterile phosphate-buffered saline (PBS). The recovered lavage fluid was centrifuged (200 g for 10 min), and red blood cells from the cellular pellet were lysed with 300 ll of ACK Lysis Buffer (Gibco). Cell counts were performed directly by optical microscopy.

Ethics statement

All experiments involving animals were carried out in compliance with French and European regulations on the care and protection of laboratory animals (European Commission Directive 86/609 andthe French Act #2001-486, issued on June 6, 2001) and performed by certified personnel. The study and all experimental protocols associated were registered and approved by the French authorities (Ministe `re de l'Enseignement Supe ´rieur et de la Recherche-Direction Ge ´ne ´rale pour la Recherche et l'Innovation-Secre ´tariat Autorisation de projet, registration number 00481.01). Animals were housed at the Lille University Animal Research Facility (De ´partement Hospitalo-Universitaire de Recherche Expe ´rimentale de Lille, France) accredited by the French Ministry of Agriculture for animal care and use in research (#B59-350009).

Paul RC Imbert et al

UBAP1 targeting by Pseudomonas TIR domain protein

The EMBO Journal

Fractionation of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains were grown in LB for 4 h and adjusted to OD600 20 in 1 ml cold 50 mM Tris-HCl pH 8.0 with 1 mM EDTA and protease inhibitors (Roche). All subsequent steps were conducted at 4°C. The cell samples were sonicated three times at 30-s intervals, with the resulting cellular debris pelleted by centrifugation three times at 4,000 g for 5 min, taking the uppermost supernatant for each spin. The total membrane fraction was separated from the soluble fraction by ultracentrifugation at 100,000 g for 1 h. After washing the membrane pellet thoroughly in sonication buffer, the inner membrane fraction was solubilized in 200 ll 50 mM Tris-HCl pH 7.6 with 2% (v/v) sodium lauroyl sarcosinate for 1 h with gentle agitation. The outer membrane fraction was pelleted by ultracentrifugation at 100,000 g for 1 h, washed and resuspended in sonication buffer. The preparation of supernatant samples separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequent immunoblotting has been described previously [START_REF] Hachani | Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins[END_REF]. Immunodetection was conducted using monoclonal antibodies against RNA polymerase (NeoClone) and polyclonal antibodies against PilQ, XcpY [START_REF] Michel | Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa[END_REF] and LasB.

Expanded View for this article is available online.

Introduction

Pathogenic bacteria produce virulence factors that usually help the pathogen to survive in an environmental niche, to promote colonization and invasion of host tissues, or to modulate the immune system. Virulence factors are toxins or effector proteins than can be transported by diverse secretion machineries in bacteria [1,2]. Once secreted, these proteins can be assembled on the bacterial cell surface, released in the extracellular space, or secreted directly into a host cell or a neighboring bacterium. Once in host cells, effectors often target key proteins to hijack the host cellular machinery to remodel signaling cascades. The yeast two-hybrid system is often used to screen a large number of host proteins that potentially interact with bacterial effectors [3]. Regarding the mechanism of the secretion systems, a bacterial two-hybrid system is frequently employed to identify interaction networks between components of the secretory apparatus, as well as interaction between effectors and proteins of the machinery [4]. However, protein-protein interactions that have been determined by twohybrid assay must be confirmed by other methods [5].

Laure Journet and Eric Cascales (eds.), Bacterial Protein Secretion Systems: Methods and Protocols, Methods in Molecular Biology, vol. 1615, DOI 10.1007/978-1-4939-7033-9_20, © Springer Science+Business Media LLC 2017

247

Pull-down is an in vitro method widely used to detect or confirm interactions among multiple proteins. This assay is similar in methodology to co-immunoprecipitation experiments in its use of an affinity ligand to capture interacting proteins. The difference between these two methods is that while co-immunoprecipitation uses immobilized antibodies to capture protein complexes, the pull-down approach uses a purified and tagged protein as a "bait" to bind any interacting proteins. The method consists of first immobilizing the tagged protein (bait) on an affinity ligand specific to the tag, creating an affinity support to capture and purify other proteins (prey) that interact with the bait. The bait and prey proteins can be obtained from multiple sources, such as cell lysates, purified proteins, expression systems, and in vitro transcription/ translation systems. Once the prey proteins have been incubated with an immobilized bait protein, interacting complexes are eluted using an eluting buffer depending on the affinity ligand. Each experiment needs proper controls to demonstrate that characterized interactions are not an artifact. For example, a positive control consisting of an immobilized bait protein alone is necessary to verify proper attachment of the tagged bait protein to the affinity support. To identify and eliminate false positives caused by nonspecific binding of prey proteins to the affinity support, cell lysates or purified proteins can be analyzed after being passed through a minus bait support. Following a pull-down experiment, protein fractions are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then visualized by gel staining or western-blotting detection.

In this chapter, we describe detailed pull-down assay procedures that allow the identification of interacting proteins. First, we focus on how to perform a pull-down experiment to identify an interaction between a bacterial bait protein and eukaryotic prey proteins expressed in host cells (Subheadings 3.1 and 3.2). Next, we present how the interaction between two purified proteins can be visualized by a pull-down assay (Subheading 3.3). In these procedures, pull-down experiments have been performed using specific bait proteins fused to a 6× histidine tag. As a consequence, we selected Ni-NTA agarose beads as the affinity support used to immobilize these recombinant proteins.

Materials

Preparation of Cell Lysate

Prepare all solutions with distilled water at room temperature and keep them at the indicated temperatures.

1. Eukaryotic cells.

2. Cell culture dish, treated for optimal cell attachment, with growth surface area around 55 cm 2 , sterile.

Methods

Preparation of Cell Lysate

Pull-Down Assay Using Cell Lysate as Prey (See Notes 10 and 11)

1. Seed eukaryotic cells at 5.10 5 in 10 cm cell culture dish (see Note 7) and incubate overnight at 37 °C in CO2.

2. Transfect cells with plasmid containing gene of interest fused to a specific tag with appropriate transfection reagent for time necessary for optimal expression of protein (16-24 h is usually a good range).

3. Cool cells by placing plates on ice, wash cells with 1× PBS. Add 2 mL cold PBS and harvest cells using cell scraper.

SDS-PAGE and Analysis of Protein Fractions

1. To 15 µL protein fraction add 5 µL Laemmli lysis buffer, 4× concentrate. Heat for 3 min at 100 °C and centrifuge 30 s using a microcentrifuge to bring down condensate.

2. Load 10 µL protein fraction and 5 µL protein ladder on SDSpolyacrylamide gel.

3. Electrophorese proteins in running buffer at 100 V for 15 min then 180 V until dye front has reached bottom of gel.

4. Identify interacting proteins by immunodetection or blue coomassie coloration (see Note 25).

Notes

1. We prefer not to use the solutions after 6 months of storage.

2. A different buffer, such as HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid), MES (2-(N-morpholino) ethanesulfonic acid), or phosphate buffers, may be required for your specific protein-protein interaction. Additionally, different pH values may be tested as these are specific and dependent on the interaction between proteins.

3. We found that pull-down experiments work better with fresh equilibrium and elution buffers.

4. The bait proteins used in this protocol are tagged with 6× His that bind the nickel agarose affinity support. The choice of the matrix-associated antibody depends on the fusion tag. The His tag is composed of a peptide motif that consists of six histidine residues with a high affinity towards metals like nickel that composes the used Ni-NTA agarose but also the Ni-NDA, Ni-TED, or Ni-TALON resins. The 6× His tag is very small (~1 kDa), which renders it less immunogenic than other larger tags, is shown not to affect the native conformation of bait proteins, and maintains its partner binding activity. Few naturally occurring proteins also bind to Ni-NTA matrices, making this tag the most commonly used affinity tag. In pull-down assays, the choice of the matrix-associated antibody depends on the fusion tag. What follow are some examples of tags with their advantages and disadvantages. The FLAG tag is an octapeptide that is likely located on the surface of the fusion protein due to the hydrophilic nature of amino acid residues and has affinity to anti-FLAG resin. Like the His tag, the FLAG tag is small, but a disadvantage is that the monoclonal antibody matrix is not as stable as Ni-NTA. Glutathione S-transferase (GST) tag binds to glutathione-associated support with high affinity and specificity. This tag has the advantage that GST isoforms are not normally found in bacteria, so purified bacterial prey proteins normally do not have affinity with glutathione resin. However, GST tag is large (26 kDa), exists as a dimer, is prone to nonspecific interaction, is expensive, and affinity to its support depends on certain reagents. The maltose-binding protein (MBP) tag from an Escherichia coli periplasmic protein has affinity for matrix consisting of sugars or anti-MBP. This tag is used for the purposes of overcoming problems associated with the expression and purification of recombinant proteins [7]. However, the disadvantage of the MBP tag is its large size, its immunogenicity, and the mild elution of MBP-tagged proteins, which complicate pull-down experiments. 5. Make an aliquot of 1 mL before -20 °C storage. This will prevent the degradation caused by repeated thawing.

6. Make an aliquot of 500 µL before -20 °C storage. The used Laemmli lysis buffer can be kept at 4 °C for 1 month.

7. As negative control, prepare a cell lysate without expressing bait protein (negative cell lysate). This will eliminate false positives resulting from nonspecific interactions of cell lysate proteins with the Ni-NTA agarose beads. Additional negative controls can include an irrelevant protein with the same tag or expression of the tag alone, as in the case of the GFP.

8. Before stocking the cells, remove an aliquot and control by western blot the production of the prey protein.

9. Whole-cell lysate instead of the supernatant fraction can also be used to test whether the prey protein of interest localizes in the pellet fraction.

10. Pull-down experiments using cell lysates will not demonstrate that interaction between the bait and prey proteins is direct but only determine that they are part of the same complex. To prove a direct interaction, the prey protein must be purified and used in pull-down experiments as described in Subheading 3.3.

11. Try to work mostly on ice or at 4 °C to prevent the degradation or the denaturation of the proteins.

12. Break the end cap of the gravity flow column and place it on a 1.5 mL Eppendorf tube. Thoroughly resuspend the Ni-NTA resin by inverting the bottle several times to obtain a uniform suspension. Pipette tips must be cut to allow the Ni-NTA agarose beads to get into.

13. This step eliminates the left 30% ethanol present in the Ni-NTA resin.

14. Before loading the bait protein, plug the gravity flow column using a piece of parafilm before replacing it on a 2 mL Eppendorf tube.

15. Prepare a supplementary column by mixing 50 µg of a known noninteracting bait fused to 6× His tag with 400 µL equilibrium buffer to an empty column. Additionally, prepare a column by adding 400 µL equilibrium buffer to an empty column. These negative bait columns will be used in combination with cell lysates to eliminate false positives resulting from nonspecific interactions.

16. The incubation time can be increased from several hours to overnight at 4 °C under agitation depending on the strength of the interaction between bait and prey proteins. 17. Rotate on roller or rotating platform.

18. The column should stand straight on the ice. This step allows the resin to flow by gravity before centrifugation.

19. We found that loading two times the flow-through increases the capacity of the binding. 22. We found that loading two times the eluted fraction increased its quantity.

23. As negative control, incubate 50 µg bait protein (minus prey) or prey protein alone (minus bait) in 400 µL equilibrium buffer. The minus prey control will ensure that the Ni-NTA agarose resin will correctly capture the His-tagged bait protein alone. The minus bait control will eliminate false positives resulting from an interaction between affinity support and prey protein.

24. A different incubation temperature and time may be required for your specific protein-protein interaction.

25. A prey protein that interacts with the bait protein will be found in the eluted fraction. In contrast, a noninteracting protein will not be retained by the bait protein, will pass through the column, and will be found in the flow-through protein fraction.

  Je remercie la Dr. Agathe Subtil et le Dr. Matteo Bonazzi d'avoir accepté d'être rapporteurs de ma thèse. Je remercie le Dr. Xavier De Bolle d'avoir accepté de juger mon travail en tant qu'examinateur. Je remercie également le Pr. Patrice Gouet d'avoir accepté de présider ce jury de thèse. Je remercie également les membres de mes différents comités de suivi de thèse, la docteure Anne Vianney et le docteur Lionel Ballut pour tous leurs conseils prodigués durant les 3 CST. Durant ma thèse, j'ai pu utiliser différents équipements de la plateforme Protein Science Facility PSF (SFR Biosciences, UMS 3444). Ainsi, je souhaite remercier pour leur accueil chaleureux et leur disponibilité : Frédéric Delolme, Aline Page, Roland Montserret, Eric Diesis et plus particulièrement Virginie Gueguen-Chaignon qui a contribué à l'obtention de la structure cristallographique. Je remercie également la plateforme d'imagerie et microscopie PLATIM (SFR Biosciences, UMS 3444/US8) et plus particulièrement Claire Lionnet.

Figure 1 .

 1 Figure 1. Global distribution of brucellosis cases in the year 2000.The most affected countries are Mongolia and the countries of the Middle East and the Balkans. Some Central and South American countries and the countries of the Mediterranean basin also have numerous cases of human brucellosis.[6]

Figure 3 .

 3 Figure 3. Schematic representation of the T4SS VirB/D. The NTPases are coloured blue, the core proteins green and the pilus yellow modified from [100]

  protein translocation reporter assay [106]. VceC interacts with Bip/Grp78, activating the Unfolded Protein Response (UPR) [108]. More recently, contradictory results have been reported related to the role of VceC. Some author found that VceC induces CHOP expression favoring ER stress in placenta trophoblast, and thus, inducing cell death and placenta inflammation, promoting abortion [109]. Other authors found that VceC inhibits the expression of CHOP protein inhibiting apoptosis mediated by ER stress and thus, protecting cell from death by apoptosis and favoring intracellular persistence [110]. Also, very recently, it has been shown that a VceA mutant promotes autophagy and inhibits apoptosis in trophoblast during Brucella infection [111]. BPE005, BPE043, BPE275 and BPE123 were identified using CyaA adenylate cyclase assay [104]. BPE123 was shown to interact with alpha enolase (ENO-1), a host cell factor involved in B abortus intracellular replication [112].

Figure 4 :

 4 Figure 4 : Location of the different classes of PRRs

Figure 5 .

 5 Figure 5. Schematic representation showing structure of TLR family. TLRs can be present at the plasma membrane of the cell but also in the endosomes present in the cell. The TIR domain of TLRs is always present in the cytosol.

90°Figure 6 .

 6 Figure 6. Cartoon representation of the horseshoe-shaped structure of human TLR3 ectodomain (2a0z), depending on the secondary structural elements: -sheet in yellow, -helices in red and loops in green. Structural representation were performed with Pymol.

  receptor [138]. Three highly conserved sequences called box 1, box 2 and box 3 have been characterized in this TIR domain after alignment of sequences from different members of the Il-1R/TLR family[139] (Figure 7A). These sequences are involved in the signal transduction downstream of the receiver. The box 1 sequence is involved in the coupling of the TLR with another receptor [140], the box 2 sequence is involved in the interaction with the adaptor molecules involved in TLR signaling [141]. The box 3 sequence contains essential amino acids NT CT NT CT for signaling and is involved in the localization of the receptor through interactions with elements of the cytoskeleton [142] and also in the interaction with the other TIR domaincontaining proteins : adaptor protein. There are 5 adaptor proteins : MyD88 (Myeloid differentiation primary response protein), TRIF (Toll/IL-1 receptor domain containing adaptor inducting IFN-β ou TICAM1 pour Toll-like receptor adaptor molecule 1), TIRAP (TIR domain containing adaptor protein ou MAL pour MyD88 adaptor-like), TRAM (TRIF-related adaptor molecule) et SARM (pour Sterile alpha-and armadillo-motif-containing protein) [126] [143].

Figure 7 .

 7 Figure 7. Sequence and strucure of TIR domains. A) Multiple sequence alignment of TIR domain proteins from Human and bacterial : Human TLR2 (residues 639-782), Human TLR4 (residues 672-815), Human TLR6 (residues 640-781), Human MyD88 (residues 159-293), Human TIRAP (residues 84-213), Human TRIF (residues 393-553), Human TRAM (residues 73-229), Brucella abortus BtpA (residues 141-275), Brucella abortus BtpB (residues 156-292), Escherichia coli TcpC (residues 169-303). Structural features above the sequence are based on the MyD88. Blue arrows denote areas of beta sheet, red boxes denote alpha helices and black line denote connecting loops. The alignment and the prediction structure were performed with PROMALS3D. B) and C) Cartoon representation of the structure of human MyD88 TIR domain (4DOM) and human TLR2 TIR domain (1FYW) respectively, depending on the secondary structural elements: -sheet in yellow, -helices in red and loops in green. D) Ribbon representation of the superposition of MyD88 TIR domain (4DOM) in cyan with TLR2 TIR domain (1FYW) with Root Mean Square Deviation (RMSD) of 2,431Å. Structural representation were performed with Pymol.

Figure 8 .

 8 Figure 8. Schematic representation of TLRs pathways. Each receptor recognizes a specific ligand. TLR1, TLR2, TLR6, TLR5, TLR11 are at the cytoplasmic membrane, while TLR3, TLR7, TLR8, TLR8 and TLR9 are endosomal.

Figure 9 :

 9 Figure 9 : The different strategies used to manipulate TLRs. (A) Microbes may stimulate other receptors that lead to a weakened immune response. (B) Microbes may modify their PAMPs in order to avoid TLR recognition. (C) Microbes may interfere with TLR signaling. Adapted from 3underhill 20043 effective in stimulating TLRs. Yersinia pestis, for example, is capable of modifying the composition of its membrane lipid A, according to the growth temperature. Thus at 37°C, host temperature, the LPS of Yersinia pestis does not stimulate the TLR4 receptor [158] [159]. In addition, Campylobacter jejuni, H. pylori and Bartonella bacilliformis produce flagellins that do not activate TLR5 [160].

  regulates the concentration of nuclear factors or serves as a hub for gene regulation. These nuclear bodies are extremely dynamic, reacting to different physiological processes and different forms of stress. They are formed at the exit of mitosis and maintained until the start of the next mitosis. Nuclear bodies are made up of proteins and RNAs whose structural

  Figure10.Schematic representation of the main nuclear subcompartments. According to3[198]

Figure 11 :

 11 Figure 11 : Schematic representation of the structure of the nucleolus consisting of a fibrillar centre, a dense fibrillar component and a granular component. According to[198]

Figure 12 .

 12 Figure 12. Schematic view of ribosome biogenesis in mammals :From the 47S precursor ribosomal RNA, a succession of treatment steps lead to the formation of the 40S and 60S subunits which will be assembled in the cytosol to form the mature 80S ribosome[316].

Figure 13 .

 13 Figure 13. Tridimentary structure of ubiquitin, SUMO1 and SUMO2. Cartoon representation of the structure of human Ubiquitin (1UBI), human SUMO1 (2N1V) and human SUMO2 (4NPN), depending on the secondary structural elements: -sheet in yellow, -helices in red and loops in green.

Figure 14 .

 14 Figure 14. Alignment of the five SUMO paralogues in humans. The preserved amino acids are in red and the poorly preserved amino acids in black. Amino acids highlighted in red are preserved in all sequences. The sequence alignment was carried out with ClustalW and ESPript3.

Figure 15 .

 15 Figure 15. Enzyme cascades involved in sumoylation . SUMO-specific proteases (SENPs) catalyse the maturation of SUMO proteins by cleaving the amino acids that follow the double glycine reside at the Cterminal end of SUMO. Mature SUMO can then be conjugated to its substrate by following a cascade of enzymatic reactions: E1 the activating enzyme (SAE1/SAE2), E2 the conjugating enzyme (Ubc9) and E3 the ligase. The substrate can be deSUMOylated by the action of the protease of the SENPs family

Figure 16 .

 16 Figure 16. Molecular consequences of modification by SUMO. SUMO and ubiquitin are indicated respectively by S and U. Adapted from [241] Created with BioRender.com

Figure 17 .

 17 Figure 17. Schematic representation of SENP family protein. Catalytic domain (CD) colored magenta, Putative SUMO-interacting motifs (SIMs) are indicated by asterisks and regions shown to be important for intracellular localization colored in cyan [231].

Figure 18 :

 18 Figure 18 : Schematic representation of the cellular distribution of the SENPs family members. The preferential distribution of SENPs is shown in green. SENP1 and SENP2 are found at the level of the nuclear envelope (NE) of the nucleoplasm, and around the PML nuclear body. SENP3 and SENP5 are found in the nucleoli (No), nucleoplasm and also in the mitochondria (Mit). SENP6 and SENP7 is only found in the nucleoplasm [250].

  Furthermore, SENP3 does not possess hydrolase activity and therefore does not participate in the maturation process of SUMO.The N-terminal part contains a domain responsible for its localization in the cell, a NoLS (Nucleolar Localization Site) domain, corresponding to a very acidic region of the protein. The absence of this domain leads to a redistribution of SENP3 from the nucleolus to the nucleoplasm. Nevertheless it seems that the localization of SENP3 is not exclusively in the nucleoli since it has been established that SENP3 may reside in the cytosol and is required for mitochondrial fission [297,298]. The biological processes in which SENP3 is involved are now well established, and include: inflammation [252], cell differentiation [253], cell stress response [297,301,302] and ribosome biogenesis [254]. Nevertheless, the identification of substrates and their physiological roles are still poorly characterized. This lack of knowledge stems in part from the difficulty of experimentally obtaining recombinant SENP3 proteins, especially the stable and active catalytic domain [255].I will now present some of the most relevant discoveries relating to SENP3 to illustrate the multiple roles of this protease in various cellular processes.1. SENP3 as a redox-sensor under stressIt has been shown that SENPs can be redox sensors[256]. Reactive oxygen species (ROS) have been shown to modulate SENP3 and have an impact on its stability and localization. Indeed, the redox state of the cell regulates the level of SENP3 expression. HeLa cells under mild oxidative stress such as treated with oxygen peroxide (H2O2) present an increased level of SENP3 due to inhibition of ubiquitination, revealing that under normal conditions SENP3 is ubiquitinated by the ubiquitin ligase CHIP (for C-terminus of Hsc70-interacting protein) and sent to the proteasome for degradation[257] [258]. Under moderate oxidative stress, two SENP3 cysteines (Cys243 and Cys274) are oxidized[259], allowing recruitment of Hsp90 and preventing ubiquitination by CHIP (Figure19)[260].

Figure 19 .

 19 Figure 19. Redox regulation of SENP3 : SENP3 interacts whith CHIP under standard and oxidative stress condition. Under oxidative condition SENP3 undergo oxidative modification which are signal for the recruitment of HSP90. Created with BioRender.com.

  [267] [268]. NPM1 is a 37kDa phosphoprotein that shuttles between the granular component of the nucleoli and the cytoplasm. The SENP3/NPM1 interaction has been shown by co-precipitation experiments in HeLa cells and by yeast two hybrid. In addition, the authors showed that NPM1 specifically interacted with SENP3, since other members of the SENPs family are not able to interact with NPM1 [269]. SENP3 catalyzes the deSUMOylation of NPM1 by removing SUMO2/3. Together SENP3 and NPM1 play a key role in ribosome biogenesis. Indeed, depletion of SENP3 or NPM1 by siRNA negatively impacts ribosome biogenesis by inhibiting the maturation of 32S rRNA into 28S [269].

a

  quality control mechanism by preventing the loading of the PELP1-WDR18-TEX10 complex on the maturing pre-ribosome[271] [272]. Thus, SENP3-mediated deSUMOylation makes it possible to coordinate the rate of ribosome formation with the physiological state of the cell.In addition, the absence of SENP3 leads to a localization error of the ribosomal transformation factor NVL (Nuclear VCP-like protein)[273]. NVL belongs to the AAA ATPase family[274]. The human genome codes for two NVL isoforms, NVL1 and NVL2, which differ in the length of their N-terminal sequence. Thus, NVL1 starts from the residue corresponding to the second methionine at position 107 of NVL2. NVL1 and NVL2 have distinct locations in the cell and more particularly in the nucleus. NVL2 is the more represented of the two, it is mostly found in the nucleoli while NVL1 is nucleoplasmic. Nagahama et al. were able to establish that NVL interacts with RPL5 in a manner dependent on ATP and their NoLS. This interaction allows NVL to have a nucleolar localization[275]. Depletion of NVL2 by siRNA inhibits ribosomal biosynthesis, highlighting that like SENP3, NVL2 plays a key role in this cellular process[275] [276]. 1. The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism When I first joined the team, the topic of my research was centered on bacterial TIR domain proteins. During this time I participated in several projects that gave rise to 4 publications. My main contribution lead to the publication included in this chapter, in which I am joint first author on the characterization of the Brucella TIR domain-containing proteins, BtpA and BtpB. In the work we show that the recently NADase activity described for TIR domains is retained in BtpA and BtpB and leads to intracellular NAD depletion during infection. This was a joint project with our long-term collaborators at the University of Madrid, the lab of Victor Cid and Maria Molina. I constructed several expression and complementation vectors as well as Brucella strains, participated in the assessment of the localization of BtpA/BtpB in transfected cells along with Paul Imbert, and carried out all the infection work, namely for the NAD measurements that were done with the help of Morgane Roussin. I also worked side-by-side on this project with Julia Coronas-Serna when she came for a 3 months stay at our lab. to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast

Fig 1 .

 1 Fig 1. Expression and localization of B. abortus BtpA and BtpB in S. cerevisiae. (A) BtpA and BtpB induce different levels of toxicity when expressed in yeast. Ten-fold serial dilution assay to monitor growth in YPH499 yeast strain expressing the pYES2 empty vector or the BtpA or BtpB indicated versions (full-length, N and TIR) from pYES2-GFP plasmid derivatives, under control (Glucose) and induction (Galactose) conditions. Nomarski and fluorescence microscopy of YPH499 yeast strain expressing from pYES2 plasmid derivatives the following fusion proteins: (B) fulllength GFP-BtpB after 4h induction; (C) full-length GFP-BtpA (green) and stained with DAPI (red), after 6h induction; (D) the GFP-fused N-terminal regions of BtpA and BtpB after 5h induction; (E) the GFP-fused C-terminal regions containing TIR domains of BtpA and BtpB after 4h induction. Scale bars correspond to 5 µm. https://doi.org/10.1371/journal.ppat.1007979.g001

Fig 2 .

 2 Fig 2. BtpB expression causes severe defects in actin cytoskeleton and endocytosis in S. cerevisiae. (A) Nomarski and fluorescence microscopy images (upper panel) and graph showing the percentage of small-to medium-budded cells with depolarized actin (lower panel) after rhodamine-phalloidin staining of YPH499 cells expressing pYES2-GFP empty vector, BtpB, BtpB-N or BtpB-TIR from pYES2-GFP plasmid derivatives after 4h induction. Data correspond to means ± standard deviation of three independent transformants (n 100) and statistical comparison was done with Kruskall-Wallis ANOVA with p-values referring to BtpB of 0.024 ( ) for vector and BtpB-N. Scale bars indicate 5 µm. (B) Nomarski and fluorescence microscopy images (left panel) and graph representing the percentage of cells showing both GFP and FM4-64 vacuolar signal (right panel) of YPH499 cells expressing pYES2-GFP or pYES2-GFP-BtpB, after 4h induction, stained with the endocytic marker FM4-64 for 1h. Data correspond to means ± standard deviation of three independent transformants (n 100) and statistical comparison was done with Student´s t-test, p < 0.0001 ( ). Scale bars indicate 5 µm. https://doi.org/10.1371/journal.ppat.1007979.g002

  Fig). The TIR domain of BtpB alone was fully responsible for signaling down-regulation (S1B Fig). Interestingly, such effect was also observed when expressing the TIR domain of BtpA (S1A Fig).

  , and it did not lead to reduced MAPK phosphorylation (S1B Fig), or endocytosis defects (S5D-S5E Fig).

Fig 3 .

 3 Fig 3. BtpB, BtpA-TIR and BtpB-TIR reduce NAD + and ATP levels when expressed in yeast. (A) Cellular ATP measurement by luciferase assay in YPH499 cells transformed with pYES2 empty vector and pYES2 plasmid derivatives bearing: both full-length and TIR domain versions of BtpA and BtpB and the catalytically inactive BtpB E234A mutant. Graph shows ATP levels as a percentage relative to the ATP levels measured on empty vector control cells. Results correspond to means ± standard deviation of three different transformants and statistical comparison was done with one-way ANOVA with p-values referred to vector of 0.0045 ( ) for BtpA-TIR, 0.0017 ( ) for BtpB and

Fig 4 .

 4 Fig 4. Functional analysis of BtpB and BtpA mutations in yeast. (A) Structure of BtpA-TIR domain showing the positions equivalent to those identified as loss-of-function in BtpB by yeast random mutagenesis screening. Left panels: two views of BtpA-TIR dimer structure (PDB: 4LZP) with chain A colored in wheat and chain B in grey. Residues identified are colored according to their assigned properties. Positions of the mutations putatively affecting protein folding are colored in blue (pA strand) and yellow (αC helix). Mutations at the active site are colored in magenta. Mutations at the surface outside the active site are colored in green. Right panels: views in the same orientation of the BtpA dimer depicted as cartoon with the side chains of mutated residues displayed as ball-and-sticks. Residue numbers are indicated for BtpA and the corresponding residues in BtpB are in parenthesis. (B) Ten-fold serial dilution growth assay of YPH499 cells expressing pYES2 empty vector, BtpB full-length, BtpB-TIR and the indicated mutants

  1 (LAMP1) (Fig 5A), suggesting BtpB associates with multiple intracellular structures. Unlike the yeast model, expression of BtpB neither resulted in significant perturbation of the actin cytoskeleton (Fig 5B) nor the microtubule network morphology (Fig 5C). Consistent with results from Felix and colleagues [19], we also observed localization of BtpB between cells, at sites of intercellular bridges that form during cell division (S6B Fig). As the yeast model revealed a potential role for the N-terminal domain of BtpB in intracellular localization whereas the TIR domain for its toxicity, we next analyzed the fate of truncated BtpB versions in HeLa cells. As in yeast, expression of BtpB-N resembled that of fulllength BtpB, with cytosolic aggregates being formed (Fig 6). Expression of the TIR domain alone (BtpB-TIR) resulted in the formation of long filament-like structures that showed no colocalization with tubulin (Fig 6), consistent with the results obtained in the yeast model. In some cells, BtpB-TIR induced disorganization of the microtubule network (S6C Fig). These filamentous structures did not co-localize with vimentin either, a marker of intermediate filaments (S6D Fig), strongly reminiscent of what has been previously described for the

Fig 5 .

 5 Fig 5. Localization of ectopically expressed BtpB in human cells. HeLa cells expressing GFP-BtpB (green) were labelled and analyzed by confocal immunofluorescence microscopy. Representative images are shown. (A) Cells were labelled with FK2 (cyan) along with LAMP1 (red) antibodies. The nuclei are labelled with DAPI (blue). (B) Cells were labelled with phalloidin for visualization of the actin cytoskeleton (red) and (C) with anti-tubulin antibody for visualization of the microtubules (red). Scale bars correspond to 5 µm. The tubulin image was obtained with Airyscan confocal imaging mode. https://doi.org/10.1371/journal.ppat.1007979.g005

  Fig 8A). As shown in Fig 8B, both BtpA and BtpB strongly reduced total NAD levels in HeLa cells validating the results obtained with the yeast model.

Fig 7 .

 7 Fig 7. Inhibition of endocytosis occurs upon ectopic expression of BtpB but not during infection. HeLa cells expressing GFP-BtpB (green) were incubated with either EGF conjugated with Alexa Fluor 555 (red) or transferrin conjugated with Alexa Fluor 568 (red) for 10 minutes. (A) Cells were then analyzed by confocal microscopy and (B) and (C) the percentage of cells showing uptake of either fluorescent marker quantified. A cell was considered positive when clear labelling of endocytic vesicles was observed throughout the cell. Counts correspond to individual microscopy fields, obtained from three independent experiments. Data correspond to means ± standard deviation and statistical comparison was done with Mann-Whitney test, p = 0.0001 ( ) for EGF (left) and p < 0.0001 ( ) for transferrin (right). (D) HeLa cells or (E) immortalized bone marrow-derived macrophages (iBMDM) were infected for

  https://doi.org/10.1371/journal.ppat.1007979.g007 fully restored the wild-type phenotype in the case of BtpA (Fig 10A) and partially in the case of BtpB (Fig 10B).

Fig 8 .

 8 Fig 8. Ectopic expression of BtpB results in reduction of total NAD + in HeLa cells. (A) Representative Western blot showing levels of Myc-BtpA and Myc-BtpB expression revealed with an anti-Myc antibody and anti-actin antibodies, as a loading control. Myc-BtpA has a predicted molecular weight of 33 kDa whereas BtpB 38 kDa. (B) Measurement of total NAD levels using a colorimetric assay from HeLa cells expressing either Myc-tagged BtpA or BtpB. Non-transfected cells (negative) and cells transfected with Myc vector alone are also included as controls. Data correspond to means ± standard deviation from three independent experiments and statistical comparison was done with one-way ANOVA, with a p-value for the negative control versus Myc-BtpA of <0.0001 ( ) and versus Myc-BtpB of 0.0006 ( ). https://doi.org/10.1371/journal.ppat.1007979.g008

Fig 10 .

 10 Fig 10. B. abortus TIR domain-containing proteins control intracellular total NAD levels during macrophage infection. (A) and (B) Immortalized bone marrow-derived macrophages (iBMDM) were infected for 48 h with either wild-type B. abortus or strains lacking btpA, btpB or both genes or complemented strains expressing btpA and btpB or the corresponding catalytic mutants. Mock infected cells are also included as a control. Total NAD levels were measured using a colorimetric assay and data correspond to means ± standard deviation from five (A) or three (B) independent experiments and statistical comparison was done with a one-way ANOVA test, with statistical significance indicated in the graph. Higher NAD levels in the infected cells than in the mock experiment are likely attributable to the fact that intracellular NAD levels from bacterial cells are added up to those of the cell line. In (A) for wild-type versus ΔbtpA p = 0.0071, wild-type versus ΔbtpApbtpA E217A p = 0.0476, ∆btpA versus ΔbtpApbtpA p = 0.0058 and ∆btpApbtpA versus ΔbtpApbtpA E217A p = 0.0398. In (B) for wild-type versus ΔbtpAbtpB p = 0.0011, wild-type versus ΔbtpB p = 0.0003, wild-type versus ΔbtpBpbtpB E217A p = 0.0001, ∆btpB versus ΔbtpBpbtpB p = 0.0156 and ∆btpBpbtpB versus ΔbtpBpbtpB E217A p = 0.0058. Statistical significances in relation to the negative control are not shown. https://doi.org/10.1371/journal.ppat.1007979.g010

  Fig. Expression of BtpB and BtpA versions in yeast and in inhibition of yeast endocytosis by the TIR domain of BtpB. Western blotting of YPH499 cells extracts bearing the indicated BtpA (A) and BtpB (B) versions from pYES2-GFP plasmid derivatives, using antibodies anti-GFP (upper panels), anti-P-MAPK to show dual phosphorylation of Slt2 yeast MAPK and anti-G6PDH as loading control (lower panels). (C) Normarski and fluorescence microscopy of YPH499 cells expressing GFP-BtpB, GFP-BtpB-N and GFP-BtpB-TIR after 4h induction, stained with the endocytic marker FM4-64 for 1h. Scale bars indicate 5 µm. (PDF) S2 Fig. BtpA and BtpB TIR filaments are not coincident with yeast tubulin. Indirect immunofluorescence of YPH499 yeast cells expressing GFP-BtpA-TIR, GFP-BtpB-TIR, and their corresponding E234A mutant versions from pYES2 plasmid derivatives (green). Microtubules are stained using anti-tubulin antibody (red). Nuclei are labelled with DAPI (blue). Scale bars correspond to 5 µm. (PDF) S3 Fig. Inhibitory effect of BtpB on phosphorylation of yeast signaling proteins.(A) Western blotting from cells bearing the empty vector pYES2 (control), BtpA or BtpB from pYES2-GFP plasmid derivatives, developed with anti-P-MAPK antibody to detect duallyphosphorylated Slt2, Kss1 and Fus3 (upper panel) and anti-actin to detect actin as loading control. (B) Upper part: representative immunoblot from yeast cell lysates bearing pYES2-GFP-BtpB (+) or pYES2 (-) and upon different conditions: 30ºC (control), high temperature (39ºC), pheromone (α-factor) or Congo red, using anti-P-MAPK (upper panel), anti-PLOS PATHOGENSBrucella TIR proteins deplete host cell NAD + Slt2 (medium panel) and anti-actin (lower panel). Lower part: densitometric measurement of WB bands corresponding to phosphorylated MAPKs Slt2, Kss1 and Fus3. The graph displays densitometric data of phosphorylated MAPKs normalized against actin and error bars show the standard deviation from three independent experiments on different transformant clones. (C) Western blotting of cells containing the pYES2 empty vector (control) or pYES2-GFP-BtpB, developed with anti-P-p38 antibody to detect MAPK Hog1 under high osmolarity. conditions (0.6M KCl). (D) Western blotting of cells expressing heterologous Akt1 (pYES3-GFP-Akt1) with either pYES2 empty vector (control) or pYES2-GFP-BtpB, using anti-P-Akt1(Thr)308 (upper panel) and anti-Akt1 antibodies. All immunoblots were performed on protein extracts from transformants of the YPH499 yeast strain after 4 h of galactose induction. (PDF) S4 Fig. Partial suppression of BtpB toxicity by overexpression of yeast genes. (A) Ten-fold serial dilution assay of yeast cells co-expressing BtpB and each of the seven suppressor ORFs isolated from a yeast genetic screen. pYES3 and pYES2 are the corresponding empty vectors for BtpB and for the overexpressed genes, respectively. (B) Western blotting of W303-1A yeast strain co-expressing GFP-BtpB and each of the proteins encoded by the suppressor genes. Antibodies anti-GFP to detect GFP-BtpB (upper panel) and Anti-G6PDH as loading control (lower panel) were used. Anti-GFP antibody allows the detection of the indicated protein Atagged proteins due to affinity of the tag with the Fc region of IgG-type antibodies. (C) and (D) Ten-fold serial dilution assays of yeast cells co-expressing BtpB-TIR (C) or BtpA-TIR (D) and the suppressor genes. pYES3 and pYES2 are the corresponding empty vectors for BtpB-or BtpA-TIR and for the overexpressed genes, respectively. (PDF) S5 Fig. Functional analyses in yeast loss-of-function mutations in conserved residues of BtpB. (A) Alignment of protein sequences of the TIR domains of BtpB, BtpA, human SARM1 and plant RUN1. Conserved residues relevant for this study are marked with the same color code as in Fig 4, except for for the catalytic site residues W213 and E217, that are colored in pink. (B) Structure of BtpA-TIR (left; PDB: 4LZP)) and RUN1-NADP+ complex (right; PDB: 6O0W), showing the equivalent positions of residues mutated in BtpB isolated in the yeast screen. Both structures cartoons are displayed in the same orientation. Side chain of mutated residues of BtpA relevant for this study are colored as in (A). The side chains of residues of the catalytic site of RUN1 are shown as ball-and-sticks and colored in pink and the NADP+ ligand is colored in cyan. Specific atoms are colored as follows: nitrogen in blue, oxygen in red and phosphorus in orange. (C) Phenotype of selected loss-of-function BtpB mutants. Ten-fold serial dilution growth assay of YPH499 cells transformed with pYES2 empty vector and pYES2 plasmid derivatives expressing full-length BtpB wild-type and mutants D158G, S162P and Y225C, under control (Glucose) and induction (Galactose) conditions. (D) Nomarski and fluorescence microscopy images of YPH499 cells expressing the GFP-BtpB indicated mutants, after 4h induction, stained with the endocytic marker FM4-64 for 1h. Scale bars indicate 5 µm. (E) Graph from the same experiment as in C representing the percentage of cells showing both GFP and FM4-64 vacuolar signal. Results correspond to means ± standard deviation of three independent transformants (n ≥ 100) and statistical comparison was done with one-way ANOVA with a p-value < 0.0001 ( ) for all four mutants versus wild-type. (PDF) S6 Fig. Localization and effects of GFP-BtpB versions in HeLa cells. (A) Representative micrograph of HeLa cells expressing Myc-BtpB revealed with an anti-Myc antibody (red) andPLOS PATHOGENSBrucella TIR proteins deplete host cell NAD +

Figure 20 .

 20 Figure 20. The Brucella NyxA and NyxB proteins are translocated 20 into host cells during infection. (A) RAW macrophage-like cells was infected for 4h with either B. abortus wild-type or virB9 15 expressing TEM1 (encoded by the bla gene) fused with NyxA, NyxB or BAB1_0466. The percentage of cells with coumarin emission, 10 which is indicative of translocation, was quantified after incubation with the CCF2-AM substrate. Data represent means ± 5 95% confidence intervals from 5 independent experiments, with more than a 500 cells counted for each condition. Kruskal-Wallis 0 with Dunn's multiple comparisons test was used. Not all statistical comparisons are shown. (B) Representative images for B. abortus wild-type or ∆virB9 carrying pbla:nyxA or pbla:nyxB to exemplify the presence of effector translocation visible in coumarin-positive cells (violet) or absence of translocation. (C) NyxA and NyxB translocation at 24h post-infection, as in A.

Figure 21 .

 21 Figure 21. Amino acid sequence alignment of NyxA and NyxB obtained with ClustalW, matrix EBLOSUM62, showing 82.1% identity, 86.6% similarity and 3.7% gaps.

Figure 22 .Figure 24 .

 2224 Figure 22. NyxA and NyxB accumulate in cytosolic and nuclear structures. (A) Accumulation of 4HAtagged NyxA (top) or 4HA-tagged NyxB (bottom) in cytosolic punctate or filament-like structures or (B) in the nucleus, in HeLa cells infected for 48h with ∆nyxA or ∆nyxB strains expressing DSRed and the corresponding 4HA-tagged effector. The cell nucleus is visible with DAPI. A fluorescence intensity profile along a defined straight line across the 4HA-positive structures is included for each image, with the HA signal represented in green and bacterial signal in red.

Figure 25 .

 25 Figure 25. NyxA and NyxB directly interact. (A) Pulldown experiment using purified NyxB against His-NyxA immobilized on a Ni NTA resin. An empty column was used as a control for non-specific binding. Interactions were visualized with coomassie blue stained gels. The flowthrough (FT), wash (W) and elution (E) fractions are shown for each sample and the molecular weights indicated (kDa). Eluted His-NyxA and NyxB are indicated with black and red arrows, respectively. (B) Microscale thermophoresis measuring the fraction of 20 nM of purified NyxA labelled with kit protein labelling RED-NHS binding to increasing concentrations of NyxB(6,67nM-219µM). Data correspond to means ± standard deviations of 3 independent experiment. The obtained Kd is indicated.

Figure 26 .

 26 Figure 26. NyxA and NyxB directly interact. (A) Pulldown experiment using purified NyxA against His-NyxB immobilized on a Ni NTA resin. Empty column was used as a control for non-specific binding. Interactions were visualized with coomassie blue stained gels. The flowthrough (FT), wash (W) and elution (E) fractions are shown for each sample and the molecular weights indicated (kDa). Eluted NyxA and His-NyxB are indicated with black and red arrows, respectively. (B) Confirmation of the identity of the two major eluted bands by mass spectrometry. The identified peptides are highlighted in green.

Figure 27 .

 27 Figure 27. Nuclear Nyx-positive structures are closely associated with PML-nuclear bodies. (A) HeLa cells were transfected for 12h with either HA-NyxA or NyxB (red) and labelled with an anti-PML antibody (green). (B) The level of co-localization measured with the Pearson's coefficient (PCC) between each effector and either PML or speckles. Co-transfection of the two effectors was used as a positive control. (C) Pull-down assay with His-NyxA or His-NyxB immobilized on Ni NTA resins that were incubated with a HeLa cell extract. Empty column was used as a control for non-specific binding.

Figure 28 .

 28 Figure 28. NyxA and NyxB are not SUMOylated in vitro. Purified GST-Ran GAP1, NyxA or NyxB were incubated with SUMO2, Ucb9 and SAE1/UBA2 in the presence or absence of ATP. GST-RanGAP418-587 It is used as a positive control, in the presence of ATP a band appears in the red frame corresponding

1

 1 11 genomic scaffold, alternate assembly HuRef SCAF_1103279188392 1 Homo sapiens chromosome 11 genomic scaffold, alternate assembly CHM1_1.0 Homo sapiens chromosome 11 genomic contig, GRCh37.p10 Primary Assembly Heparan sulfate proteoglycan 2 (HSPG2) 1 Homo sapiens RNA binding motif, single stranded interacting protein 3 (RBMS3) Homo sapiens alkaline phosphatase, intestinal (ALPI), mRNA 1 Homo sapiens alkaline phosphatase, placental-like 2 (ALPPL2), mRNA Homo sapiens alkaline phosphatase, placental (ALPP), mRNA SENP3 belongs to a family of cysteine proteases that share a conserved catalytic domain, characterized by a papain-like fold [231]. The variable N-terminal region often contributes to intracellular targeting of the protease. In the case of SENP3, the N-terminal region is implicated in nucleolar targeting, encoding a nucleolar localization sequence (NoLS) and phosphorylation site for mTOR, necessary for subsequent interaction with NPM1 and nucleolar targeting [268] (Figure 29A).

Figure 29 .

 29 Figure 29. The Nyx effectors interact with host protease SENP3. (A) Schematic representation of SENP3, highlighting its catalytic domain and its N-terminal nucleolar localization sequences (NoLS). (B) Pull-down assay with the N-terminal region of SENP3 from amino acid 7 until 159 (SENP37-159) against His-V5-NyxA or His-V5-NyxB immobilized on Ni NTA resins. An empty column was used as a control for non-specific binding and purified His-NyxA and His-Nyx-B inputs are shown. Interactions were visualized by western blotting using anti-SENP3 antibody, and column binding with anti-V5 (lower blot). Non-bound fractions (F1 and F2), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa).

Figure 30 .

 30 Figure 30. SENP3 is important for efficient B. abortus intracellular multiplication. (A) Western blot of HeLa cell lysate treated with siRNA control (siControl) or siRNA SENP3 (siSENP3) for 72h. Membrane was probed with an anti-SENP3 antibody followed by anti-actin for loading control. (B) Depletion was also verified by microscopy, showing a predominant nucleolar localization of SENP3 in control cells which is strongly reduced in siSENP3 treated cells. Scale bar is 5 m. (C) Bacterial colony forming units (CFU) counts of wild-type B. abortus following 2, 24 or 48h of infection of HeLa cells pre-treated with either siControl or siSENP3 for 72h. Data correspond to means  95% confidence intervals from 3 independent experiments. A two-way ANOVA with Bonferroni correction was used to compare siControl and siSENP3 at each time-point. (D) HeLa cells depleted for SENP3 or treated with the control

Figure 31 .

 31 Figure 31. The NyxB structure defines a novel family of effectors and allowed identification of the SENP3 interacting groove. (A) Two views of the NyxB monomer depicted in ribbon with helices coloured in wheat, strands in blue and loops in pink. (B) Two views of the NyxB dimer. (C) Structurebased sequence alignment of NyxB and NyxA. Secondary structure elements are indicated above the sequences. Identical residues are not shaded, residues shaded in black and grey are non-conserved

Figure 32 .

 32 Figure 32. NyxA and NyxB dimer formation. (A) Chromatograms (A280, plain) and mass measurements (dots) by Multi Angle Light Scattering of NyxB (left, green) and NyxA (right, blue). The average molecular weight determined is indicated. (B) Surface representation of dimer A (chains A and H) and

Figure 33 .

 33 Figure 33. Identification of the SENP3 interacting groove. (A) Surface representation of NyxB dimer coloured according to electrostatic potential (red negative, blue positive) showing the extended acidic patch.The inset shows a close-up view of the area with residues' side chains displayed as ball-andsticks and mutated residues (E82,Y66 and D80) coloured in cyan. (B) Pull-down assay with His-NyxA, His-NyxB or the specific catalytic mutants (His-NyxA MAG or His-NyxB MAG ) immobilized on Ni NTA resins that were incubated with a HeLa cell extract. Empty column was used as a control for non-specific binding. Interactions with endogenous SENP3, NPM1 or Histone 3 (H3) were visualized by western blotting using the corresponding antibody, and column binding with anti-His (lower blot). Non-bound fractions (F1 and F2), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa). The cell extract and the different purified Nyx inputs are also shown.

Figure 34 .

 34 Figure 34. The Brucella Nyx effectors directly reduce the SENP3 nucleolar localization in host cells. (A) Representative confocal microscopy images of HeLa cells expressing the HA empty vector (top),

Figure 35 .

 35 Figure 35. The Brucella NyxA effector directly reduces the SENP3 nucleolar localization in host cells. (A) Representative confocal microscopy images of cells expressing HA-NyxB (top) and HA-NyxB MAG (bottom), with nucleolin (red), SENP3 (white) and HA (green). (B) Quantification of the Pearson's coefficient of SENP3 versus nucleolin in HeLa cells infected for 48h with either B. abortus wild-type or

Figure 36 .

 36 Figure 36. The Brucella Nyx effectors with mutated acidic grooves are still translocated during infection. RAW macrophage-like cells was infected for 24h with B. abortus wild-type expressing TEM1 (encoded by the bla gene) fused with NyxA, NyxA MAG , NyxB or NyxB MAG . The percentage of cells with coumarin emission, which is indicative of translocation, was quantified after incubation with the CCF2-AM substrate. Data represent means ± 95% confidence intervals from 5 independent experiments.

Figure 37 .

 37 Figure 37. The Brucella NyxB effector also potentially contributes to SENP3 mislocalization during infection. (A) Quantification of the Pearson's coefficient of SENP3 versus nucleolin in HeLa cells infected for 48h with either B. abortus wild-type or nyxB, its complemented strain nyxB::Tn7-nyxB or double deletion mutant nyxAnyxB. Data are represented as means  95% confidence intervals from 4 independent experiments. (B) Representative confocal microscopy images of HeLa cells infected with the different strains expressing DSRed and labelled for SENP3 (green), in comparison to mock infected control cells (non-infected first panel).

Figure 38 .

 38 Figure 38. B. abortus does not induce significant delocalization of PES1 and NPM1 from the host nucleoli. (A) Representative confocal images of HeLa cells infected for 48h with wild-type B. abortus and labelled for DNA to visualize bacteria and cell nuclei (cyan) and PES1 (yellow). Infected cells are indicated with an asterisk. (B) HeLa cells infected for 48h with DSRed-expressing wild-type B. abortus and labelled for NPM1 (white). All scale bars correspond to 5 m.

Figure 39 .

 39 Figure 39. B. abortus induces cytoplasmic punctate accumulation of NVL in a manner partially dependent of the Nyx effectors. (A) Confocal microscopy image of wild-type B. abortus (expressing DSRed) infected HeLa cells labeled with an anti-NVL antibody (white). (B) Quantification of the number of NVL-positive cytosolic structures in mock infected control cells in comparison to wild-type or a mutant strain lacking both nyxA and nyxB. Data are represented as means  95% confidence intervals

Figure 40 .Figure 41 .

 4041 Figure 40. Deletion of NyxA and NyxB does not impact intracellular multiplication of B. abortus. Enumeration of bacterial colony forming units (CFU) of wild-type B. abortus, virB9, nyxA, nyxB or nyxAnyxB following 2, 24 or 48h of infection of HeLa cells. Data correspond to means  95% confidence intervals from 3 independent experiments.

  selected another intracellular pathogen, Legionella pneumophila, that multiplies in an ERderived vacuole inducing ER stress as observed for B. abortus[283]. Experiments done by Monica Rolando in the team of Carmen Buchrieser (Inst. Pasteur) showed tha infection with L. pneumophila did not result in NVL cytosolic accumulation (Figure42), suggesting this phenomenon is specifically induced during B. abortus infection.

Figure 42 .

 42 Figure 42. Legionella infection does not induce NVL cytosolic accumulation. Immunofluorescence analysis of THP-1 cells infected 4 and 24 hours with wild type L. pneumophila strain Paris, carrying a DsRed expressing plasmid. Cells were stained with an anti-NVL antibody (green) and analyzed by confocal microscopy. DAPI, light blue; Phalloidin, grey. Scale bars correspond to 10 µm.

Figure 43 .

 43 Figure 43. B. abortus induces cytosolic accumulation of RPL5. Representative confocal microscopy images of (A) HeLa cells or (B) iBMDM infected with wild-type DSRed-expressing B. abortus for 48h and labelled for RPL5 (white) and DAPI (blue). Zoomed cells are included on the right.

Figure 44 .Figure 45 .Figure 46 .

 444546 Figure 44. B. abortus NVL-punctate cytosolic structures are enriched in Nyx effectors. Representative confocal microscopy images of (A) HeLa cells or (B) iBMDM infected with B. abortus strains expressing either 4HA-NyxA or NyxB (green) for 48h and labelled for NVL (white) and DAPI (blue). Scale bars correspond to 5 µm.

Figure 47 .

 47 Figure 47. NVL punctate cytosolic structures are enriched in Nyx effectors and the ribophagy receptor NUFIP1. Representative confocal images of HeLa cells infected for 48h with (A) infected with wild-type

Figure 48 .

 48 Figure 48. NVL, Nyx and NUFIP1-positive cytosolic punctate structures are not lysosomal compartments. Representative confocal microscopy images of HeLa cells infected for 48h with DSRedexpressing (A and C) wild-type B. abortus or (B) nyxA encoding 4HA-NyxA. Cells were labelled for DAPI (cyan), the lysosomal marker LAMP1 (green) and either (A) NVL, (B) HA or (C) NUFIP1 (white). In (C)

Figure 49 .

 49 Figure 49. NVL-positive cytosolic punctate structures are observed under starvation condition. Representative confocal microscopy images of HeLa cells untreated or treated with HBSS (starved cells) for 4h and labelled with NVL (white). All scale bars correspond to 5µm.

3 .

 3 Brucella expressing vectors DNA fragments coding for NyxA and NyxB were obtained by PCR amplification from the B. abortus 2308 genome, digested with XbaI and cloned into pFlagTEM1 [290] using Infusion (Takara Bio). After verification by sequencing, plasmids were introduced into B. abortus 2308 or virB9 by electroporation. B. abortus 2308 knockout mutant ΔnyxA and nyxB were generated by allelic replacement. Briefly, upstream and downstream regions of about 750 bp flanking each gene were amplified by PCR (Q5 NEB) from the B. abortus 2308 genomic DNA. An overlapping PCR was used to associate the two PCR products and the ΔnyxA or ΔnyxB fragments were digested and cloned

  -acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract broth or on ACES-buffered charcoal-yeast (BCYE) extract agar. Human monocyte cell line (THP-1) was cultured and infected as previously described[291]. For immunofluorescence analyses cells were fixed in 4 % paraformaldehyde, permeabilized with PBS-triton 0.5% and stained with 4-6-diamidino-2-phenylindole (DAPI), Phalloidin and and primary anti-NVL antibody (16970-1-AP). Immunosignals were analyzed with a Leica SP8 microscope at 63X. Images were processed using ImageJ software. 9. Pulldown assays 50 µg of His tag recombinant protein was incubated with recombinant protein during 2 h at 4°C, then incubated within gravity flow column (Agilent) containing 80 µl Ni-NTA agarose beads (Macherey-Nagel) during 1 h beforehand washed in water and pre-equilibrated in equilibrium buffer 20 mM Tris-HCl pH7.5, 250 mM NaCl. The column was washed successively three times in equilibrium buffer supplemented with 25 mM imidazole, three times in equilibrium buffer and eluted in equilibrium buffer supplemented with 500 mM imidazole.

10 .

 10 Quantification of SENP3 localizationColocalization analysis for the transfected HeLa cells were performed with a custom ImageJref1/Fijiref2-based macro, that segmented the nuclei and the nucleoli of the cells in each image, classified the cells in two classes according to the intensity of HA-NyxA/NyxB, and then measured in the areas of each nucleus the Pearson correlation coefficients (by calling the plugin Coloc2 -https://github.com/fiji/Colocalisation_Analysis) between the signal of SENP3

  fluorescence. In practice, one of the partners is labelled with a fluorescent molecule or is capable of fluorescing natively. It is put in contact with a ligand at 10-16 different concentrations and then placed in capillaries. This system makes it possible to measure the dissociation constant of the interaction between two molecules without immobilization of a partner. It is a technique requiring little biological material and allows the study of interactions from nanomolar to milimolar. The experiments were performed at 20°C on a Monolith NT.115 (Nanotemper) with Premium Coated (Nanotemper) capillaries. The tested interactions were performed in triplica. The labelling of NyxA is performed by covalent coupling of the lysines of the protein to a fluorophore using the RED-NHS (Nanotemper) Protein Labeling Kit. Unlabeled

14 .

 14 Protein expression and purificationE. coli BL21-DE3-pLysS bacteria were transformed with the expression vectors, grown in LB media (Sigma-Aldrich) to OD280=0.6 and expression was induced with 1mM IPTG at 37°C for 3 hours. Cells were After harvested by 15 min of centrifugation at 5,000 g and resuspended in lysis buffer 20mM Tris pH 8, 150mM NaCl, 5% Glycerol, 0,1% Triton. Disruption cell was achieved with sonication after addition of antiprotease EDTA-Free cocktail (Roche) and 30U/ml benzonase (Sigma-Aldrich). Cell debris were removed by centrifugation 30 min at 20,000g at 4°C. Recombinant protein was purified by chromatography using a Nickel-loaded Hitrap Chelating HP column (GE Healthcare). Unbound material was extensively washed using Tris 20mM pH8, NaCl 300mM, 25 mM Imidazole, 5mM -mercaptoethanol, 10% Glycerol. An additional washing step with 2 column volumes of 1M NaCl was done before elution of NyxB over a 25 to 500 mM gradient of imidazole over 8 column volumes. Peak fractions were pooled and the His tag was cleaved with TEV protease (500 µg/20mg of eluted protein) in presence of 1 mM DTT and 0,5 mM EDTA in overnight dialysis buffer 20mM Tris pH 8, 150mM NaCl.NyxB was further purified by size exclusion chromatography (Superdex 200 HiLoad 16/600, GE Healthcare) equilibrated in 20mM Tris pH 8, 150mM NaCl, 5 % Glycerol. Purity of the sample was assessed by SDS-PAGE. Freshly purified NyxB was concentrated to 21 mg/ml on 3 KDa Amicon Ultra concentrators (Millipore). SeMet-NyxB was produced in M9 minimum medium and purified as above, to a final concentration of pure SeMet-NyxB of 24 mg/ml.15. Crystallization and data collectionScreening was conducted using a Mosquito workstation (TTP Labtech) on commercial crystallization solutions with the sitting-drop vapour diffusion technique, against a protein solution. All crystallization trials were performed at 19°C and visualized on RockImager 182 (Formulatrix). Crystals of native NyxB were obtained with 21 mg/ml NyxB in 25% PEG4000, 0,2M CaCl2, 100 mM Tris pH 8. Crystals were frozen in reservoir solution supplemented with 15% Gly. Diffraction data was collected at the European Synchrotron Radiation Facility (ESRF) Beamline line ID23EH1 and crystals diffracted up to 2.5 Å resolution in space group P6122, with 12 molecules per asymmetric unit. Crystals of Se-Met NyxB were obtained using a reservoir solution containing 2.6 M NaFormate and crystals were cryoprotected in 2.8 M Naformate supplemented with 10% glycerol. Data were collected at SOLEIL on beamline Proxima-2 from a single crystal that diffracted up to 3.7 Å resolution and belonged to the space group P6222, with 2 molecules per asymmetric unit. Diffraction data were processed using XDS[292] and with SCALA from the CCP4 program suite[293].The structure was solved using the single anomalous dispersion method on Se-Met crystals usingAutoSol [294] from the Phenix program suite[295]. An excellent experimental electron density map enabled us to manually build an initial model. The resulting model was then used for molecular replacement with data from native NyxB crystal using Phaser[295].Twelve monomers were positioned and the resulting electron density map was then subjected to the AutoBuild program, part of the Phenix program suite[295]. Model building were completed with sessions of manual model building using Coot combined with model refinement using Phenix[START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. The final model was refined to a final Rwork/Rfree of 0.20/0.24 with excellent geometry. The coordinates and structure factors of NyxB have been deposited in the protein DataBank with the code 7AD4. Figures were generated with

Pymol. 16 .

 16 Size exclusion Multi-angle light scatteringSize exclusion chromatography (SEC) experiments coupled to multi-angle laser light scattering (MALS) and refractometry (RI) were performed on a Superdex S200 10/300 GL increase (GE Healthcare) for NyxA and a Superdex S75 10/300 GL (GE Healthcare) for NyxB. Experiments were performed in buffer 20 mM Tris pH 8, 150 mM NaCl, 5 % Glycerol. 100 μl of proteins were injected at a concentration of 10 mg.ml -1 . On-line MALS detection was performed with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses (Mw) were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA). 17. Small-angle X-ray scattering SAXS data were collected for NyxA and NyxB on BioSAXS beamline BM29, ESRF using an online size-exclusion chromatography setup. 50 µl of protein (10 mg.ml -1 ) were injected into a sizeexclusion column (Agilent BioSec-3) equilibrated in 50 mM Tris, pH 8.0, 200 mM NaCl. Images were acquired every second for the duration of the size-exclusion run. Buffer subtraction was performed by averaging 20 frames either side of the peak. Data reduction and analysis was performed using the BsxCuBE data collection software and the ATSAS package[296]. The program AutoGNOM was used to generate the pair distribution function (P(r)) and to determine Dmax and Rg from the scattering curves (I(q) versus q) in an automatic, unbiased manner. Theoretical curves from the models were generated by FoXS[297]. Ab initio modelling was performed with GASBOR[298].

Figure 51 :

 51 Figure 51 : NyxA and NyxB are targeted to the yeast nucleus in an alpha importin-dependent manner. A) Transformants of YPH499 strain with either pYES2-GFP-NyxA or pYES2-GFP-NyxB, as indicated, were incubated in synthetic galactose medium for 6 h, stained with DAPI and observed. Cells expressing GFP-NyxA/B fusion proteins had a signal coincident with DAPI-stained nuclei. B) Srp1 function is required for nuclear localization of NyxA. Transformants of the W303 (SRP1) or thermosensitive isogenic JCY1410 (srp1-31) strains with pYES2-GFP-NyxA were incubated for 6h at 24 ˚C or shifted for 1 h to 37 ˚C after 5 h of incubation, as indicated. Cells with nuclear fluorescence in the population were counted (n > 100) and expressed as percentage. Data are the average of three individual transformant clones. Bars indicate the standard deviation. A student t-test was performed for statistical significance. Data from Victor Cid's lab.

Figure 52 :

 52 Figure 52 : Illustration of the potential assembly between NyxA and NyxB

Figure 53 :

 53 Figure 53 : Ectopically expressed SerB and BspJ are associated with PML-nuclear bodies like Nyx. HeLa cells were transfected 18h with either HA-BspJ, HA-SerB or co-transfected with HA-SerB and Myc-NyxB and labelled with anti-PML antibody (green). All microscopy images displayed have scale bars corresponding to 5 m. Images from Emilie Mills, Erasmus student in the lab.

  structures. A study published in the summer of 2020 presented for the first time NVL outside the nucleus, in the cytosol. The authors established that NVL was involved in the absorption of albumin. Absorption of albumin involves a multi-ligand receptor complex called CUBAM containing cubulin (CBN) and amnionless (AMN). Although the mechanisms of albumin endocytosis are not fully characterized, the authors observed that NVL interacts with AMN.They observed that the co-expression of NVL and AMN in cells led to the delocalization of NVL from nucleoli to a cytosolic compartment and that the abolition of NVL expression greatly altered albumin internalization, revealing a new function of NVL in endocytic regulation[305].

Figure 54 :

 54 Figure 54 : General schema of autophagy.[306]

1 .

 1 Annexe 1: The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility Patot et al PLoS Pathogens 2016

Table 1 .

 1 Distribution of tirS gene among MSSA and MRSA clinical strains. Staphylococcus aureus TIR Protein Modulates Host Virulence CC/ST ab SCCmec bc Clone name b No. of strains tested No. of positive PCR tests for tirS /no. of strains tested (

Table 1 .a

 1 (Continued ) Staphylococcus aureus TIR Protein Modulates Host Virulence CC/ST ab SCCmec bc Clone name b No. of strains tested No. of positive PCR tests for tirS /no. of strains tested (CC: clonal complex; ST: sequence type b CC/ST, SCCmec, and MRSA clone name were identified using the identibac S. aureus Genotyping Kit. c -: absence of SCCmec d tirS gene detected by specific PCR as described in the Material and Methods * SCCmec element sequencing doi:10.1371/journal.ppat.1006092.t001

Fig 1 .

 1 Fig 1. Characterization of the genomic context of the tirS region in S. aureus MRSA and MSSA strains. Localization of the tirS gene and surrounding context in 4 tirS-positive MRSA strains and 2 tirS-positive MSSA strains. For all 6 strains, the tirS gene was found within the SCC element. Schematic diagrams represent the genomic comparison of the 6 SCC regions of both MRSA and MSSA strains used in our study and the SCC476 element of strain MSSA476 for which the tirS gene was first described [9]. Predicted ORFs are marked in the direction of transcription as arrows. The highly conserved tirS region, consisting of the tirS gene and 4 surrounding genes among which we find the fusC gene (fusidic acid resistance), is represented by green arrows. Light rose arrows represent genes coding for antibiotic resistance, orange arrows represent the mecA gene conferring resistance to beta-lactams and blue-gray arrows represent the site specific recombinases (ccrAB or ccrC) of SCC elements. The SCC elements ISS(L) and ISS(L)) are also represented as black vertical bars. Abbreviations: ccrAB = cassette chromosome recombinase A and B; fusC = fusidic acid resistance gene; mecA = methicillin resistance gene; ccrC: cassette chromosome recombinase C; ble = bleomycin resistance gene; aaD/knt = kanamycin nucleotidyltransferase (resistance gene).

Fig 3 .

 3 Fig 3. TirS interferes with TLR and IL-1R signaling. (A) HEK293T cells were transiently transfected for 24 h with the luciferase reporter vector and murine TLR2, TLR4, TLR5, or TLR9 in the presence or absence of TirS (50 ng). Cells were then stimulated with the appropriate ligand (PAM, LPS, Fl-ST, and CpG) for 6 h before measurement of luciferase activity. White bars correspond to negative control, black bars to cells stimulated with the appropriate ligand, and gray bars to cells transfected with TirS and stimulated with the ligand. Data represent the means ± SEM of the relative luciferase activity and were obtained from duplicates of 3 independent experiments. (B) Luciferase activity of murine TLR2 following transfection with

Fig 4 .Fig 5 .

 45 Fig 4. S. aureus deleted for tirS induced larger skin lesions in WT C57Bl/6 mice. (A) Data are presented as mean total lesion size (mm 2 ) ± SEM and are representative of 3 independent experiments with at least 4 mice/group. * p < 0.05; ** p < 0.01 (B) Photographs of representative lesions at day 7 after staphylococcal infection. Black bars indicate 10 mm. Abbreviations: WT = wild-type; ΔtirS = deleted for the tirS gene; ΔtirS + tirS = ΔtirS strain chromosomally restored for tirS gene. doi:10.1371/journal.ppat.1006092.g004

Fig 6 .

 6 Fig 6. S. aureus deleted for tirS induced similar skin lesion size in MyD88-deficient mice. Data are presented as mean total lesion size (mm 2 ) ± SEM and are representative of 2 independent experiments with at least 4 mice/group. Abbreviations: WT = wild-type; ΔtirS = deleted for the tirS gene. doi:10.1371/journal.ppat.1006092.g006

  All mouse protocols were carried out in strict accordance with the Directive 2010/63/EU revising Directive 86/609/EEC on the protection of animals used for scientific purposes. This directive was translated in the French regulation as the De ´cret N˚2013-118 of February 2013 under the jurisdiction of the Ministry of Education, Research and Technology. The initial research project had been approved by the local Animal Ethic Evaluation Committee CEC-CAPP (Comite ´ d'Evaluation Commun au Centre Le ´on Be ´rard, à l'Animalerie de transit de l'ENS, au PBES et au laboratoire P4) with the references ENS_2014_025 and ENS_2014_052 and subsequently authorized by the French Ministry of Education, Research and Technology.

2 .A

 2 Annexe 2 : A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors Imbert et al. EMBO Journal 2017 This work identified PumA, a TIR protein of an atypical P. aeruginosa strain, as a key virulence determinant capable of interfering with both TLR and TNF receptor signalling. This work also implicated for the first time a role for the endosomal-sorting complex required for transport I (ESCRT-I) in modulation of intracellular trafficking of TLR adaptors. I carried out all the in vitro interaction assays with purified PumA and PumA 1-136 either using eukaryotic cell extracts expressing TIRAP and Myd88 or by co-expression in E. coli and realized lipid binding experiments. Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors Paul RC Imbert 1 , Arthur Louche 1 , Jean-Baptiste Luizet 1 , Teddy Grandjean 2 , Sarah Bigot 1 , Thomas E Wood 3 , Stéphanie Gagné 1 , Amandine Blanco 1 , Lydia Wunderley 4 , Laurent Terradot 1 , Philip Woodman 4 , Steve Garvis 5 , Alain Filloux 3 , Benoit Guery 2 & Suzana P Salcedo 1,*

Figure 1 .Figure 2 .

 12 Figure 1. PumA is required for Pseudomonas aeruginosa PA7 virulence in vivo. A Caenorhabditis elegans survival curve. Fifty C. elegans were infected with E. coli OP50 and with highly virulent strain P. aeruginosa PA14. One hundred C. elegans were infected with P. aeruginosa PA7 and PA7 ∆pumA. Test of Mantel-Cox was used with ***P = 0.0002. B To establish an in vivo model of acute infection, mice were intranasally infected with 4 × 10 7 CFU P. aeruginosa PA7 or PA7DpumA strains (n = 7/group). Lethality was monitored for 96 h, and a test of Mantel-Cox was used, with **P = 0.0035. C-E Mice were intranasally infected with 3 × 10 7 CFU P. aeruginosa PA7 or PA7DpumA strains (n = 7/group). Cells from bronchoalveolar lavage (BAL) were counted (C). Bacterial load in the lungs (D) and dissemination (E) were assessed through cultured lung or spleen homogenate. Non -parametric two-tailed Mann-Whitney test was carried out with (C) *P = 0.0173, (D) *P = 00364 and (E) P = 0.3629. All data correspond to mean ! standard error.

  ). PumA enrichment was observed with PumA tagged with Myc or HA and MyD88 fused to either HA, FLAG or Myc. Curiously, this phenotype was exacerbated when GFP-PumA, normally at the cell surface (Fig EV3) was co-expressed with MyD88, resulting in the majority of GFP-PumA being recruited to MyD88-positive compartments (Fig EV3C).

Figure 3 .Figure 4 .

 34 Figure 3. PumA co-localizes with TIRAP at the plasma membrane and to a lesser extent with intracellular MyD88, when ectopically expressed in host cells. A, B Confocal microscopy of HeLa cells (A) and mouse embryonic fibroblast (MEFs) (B) co-expressing Myc-PumA and adaptor proteins HA-TIRAP (top panel) and HA-MyD88 (bottom panel). Cells were fixed after 10 h of transfection. Scale bars correspond to 10 lm. C, D Representative micrographs obtained by super resolution structure illumination microscopy (SIM) of MEFs co-expressing (C) Myc-PumA and TIRAP and (D) Myc-PumA and HA-MyD88. Wide field (WF) is shown in top panels and structured illumination of wide field (SIM) in bottom panels. Scale bars correspond to 1 lm.

HA

  therefore carried out co-IP experiments with GFP-PumA137-303 expressed in host cells. We could not detect any interaction between nor between. However, it is important to note that expression of PumA137-303 results in loss of plasma membrane localization. Instead, we observed formation of cellular aggregates that are positive for FK2 labelling(Fig EV4C), which recognizes mono-and poly-ubiquitinated proteins and could correspond to misfolded protein. For this reason, we cannot completely exclude a role of the C-terminus of PumA in these interactions. Nonetheless, our data identify TIRAP and MyD88 as host cell targets

  EMBO Journal Vol 36 | No 13 | 2017 ª 2017 The Authors

Figure 5 .

 5 Figure 5. PumA is also capable of interacting with MyD88. A Pull-down assay using extracts from cells expressing HA-MyD88 against His-PumA or His-PumA1-136 immobilized on a Ni-NTA resin. Empty column was used as a control for non-specific binding. Interactions were visualized by Western blotting using anti-HA antibody, and column binding with anti-His (lower blot). Nonbound fraction (FT), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa). B Co-immunoprecipitation (co-IP) assay from cells expressing GFP-PumA and either Myc-TIRAP or Myc-MyD88. GFP was used as a control for non-specific binding. The co-IP was revealed using an anti-Myc antibody, the fraction bound to GFP-trapping beads using an anti-GFP antibody and the inputs (shown on the bottom two images) using both anti-Myc and anti-GFP antibodies. C-E Co-purification of His-PumA1-136 co-expressed in E. coli BL21 with either (C) His-MBP (control), (D) His-MBP-TIRAP or (E) His-MBP-MyD88. Interactions were visualized with coomassie blue stained gels. Soluble fraction (SF) and selected elutions (E) are shown for each sample and the molecular weights indicated (kDa).Source data are available online for this figure.

  which is not dependent on TIR-TIR interactions(Fig EV5A). TNFR1 inhibition was specific to PumA as expression of the Brucella TIR domain-containing protein BtpA/TcpB did not have any significant effect(Fig EV5B). We therefore carried a yeast two-hybrid screen to identify an alternative target of PumA and found UBAP1, a key component of the ESCRT-I mediating trafficking and sorting of ubiquitinated cargo proteins on MVBs[START_REF] Stefani | UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting[END_REF][START_REF] Agromayor | HA-MyD88 Myc-PumA HA-MyD88 Myc-PumA Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 Myc-PumA HA-TIRAP Zoom Myc-PumA HA-TIRAP Myc-PumA HA-MyD88 Zoom Myc-PumA HA-MyD88 References[END_REF]. This interaction was confirmed by co-IP from cells coexpressingPumA and UBAP1 (Fig 6A and B) as well as by pulldown using purified PumA or PumA1-136 and cell extracts with either streptavidin-tagged UBAP1 (Fig 6C) or Myc-UBAP1 (Appendix Fig S5A). These results were specific to PumA as the Brucella TIR protein BtpA/TcpB did not show any interaction (Fig 6C). Furthermore, PumA137-303 could not co-IP UBAP1 (Fig EV4D) supporting a role of the TIR domain in targeting UBAP1. Not surprisingly, microscopy analysis of cells expressing both PumA and UBAP1 showed significant co-localization at the plasma membrane and intracellular compartments (Appendix Fig S5C).We next investigated whether PumA is interacting with UBAP1 in the context of the ESCRT-I machinery. As over-expression of UBAP1 could result in its mislocalization, we carried out endogenous co-IP from cells expressing HA-PumA. Full-length PumA not only interacted very efficiently with endogenous UBAP1 but more importantly also co-immunoprecipitated TSG101 (Fig 6D), confirming PumA is targeting the ESCRT-I machinery. As expected, PumA also interacted with endogenous TIRAP (Fig 6D). The TIR domain of PumA only weakly interacted with endogenous UBAP1 and TIRAP (Fig 6E), whereas the C-terminus of PumA showed no interactions (Fig 6F).

Figure 6 .

 6 Figure 6. Identification of UBAP1 as a novel host protein targeted by the bacterial TIR domain of PumA. A, B Co-immunoprecipitation (co-IP) assay from cells expressing Myc-UBAP1 (A) or Strep-UBAP1 (B) with either GFP, GFP-PumA or TIRAP-GFP. The co-IPs were revealed using an anti-Myc (A) or anti-UBAP1 (B) antibodies, the fractions bound to GFP-trapping beads using an anti-GFP antibody and the inputs using anti-Myc, anti-GFP or anti-UBPA1 antibodies as indicated. C Pull-down assay using extracts from cells expressing Strep-UBAP1 against His-PumA or His-PumA1-136 immobilized on a Ni-NTA resin. Empty column was used as a control for non-specific binding. Interactions were visualized by Western blotting using anti-UBAP1 antibody, and column binding with anti-His (middle blot), followed by anti-V5 (lower blot), necessary for detection of BtpA, which for reasons we do not understand cannot be easily detected with the anti-His antibody (Appendix Fig S5B). D-F Endogenous co-IP from cells expressing (D) HA-PumA, (E) HA-PumA1-136 and (F) HA-PumA137-303. The fractions bound to HA-trapping beads were probed with anti-HA, anti-UBAP1, anti-TIRAP and anti-TSG101 antibodies. Non-bound fraction (FT), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa).Source data are available online for this figure.

  Figure 7.

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  TheEMBO Journal Vol 36 | No 13 | 2017 ª 2017 The Authors

Figure 7 .

 7 Figure 7. Analysis of the impact of UBAP1 on TIRAP and MyD88. A Representative micrographs obtained by confocal microscopy of HeLa cells co-expressing Myc-UBAP1 (red) and adaptor proteins HA-TIRAP (green, top panel) or HA-MyD88 (green, bottom panel). Cells were fixed after 10 h of transfection. Scale bars correspond to 10 lm. B Different zoomed images showing HA-MyD88 (green) recruitment to the plasma membrane in the presence of Myc-UBAP1 (red). Scale bars correspond to 10 lm. C Quantification of plasma membrane localization of MyD88 in cells expressing MyD88 alone or with either UBAP1, TIRAP or PumA. At least 200 cells were enumerated in three independent experiments, and membrane localization was defined under the strict criteria of clear line at the plasma membrane. Cells with MyD88-positive vesicles in close proximity to the plasma membrane were not counted as positive. Non -parametric one-way ANOVA Kruskal-Wallis test was performed, with Dunn's multiple comparisons test. **P < 0.01. D, E Endogenous co-IP from cells expressing (D) HA-MyD88 and (E) HA-TIRAP. The fractions bound to HA-trapping beads were probed with anti-HA, anti-UBAP1, anti-TIRAP and anti-TSG101 antibodies. Non-bound fraction (FT), last wash (W) and elution (E) are shown for each sample and the molecular weights indicated (kDa). FWestern blot of TNFR1 in A549 cells infected for 1 h with either P. aeruginosa PA7 wt, ∆pumA or ∆pumA:pumA (Ara) induced with 1% arabinose. A mock-infected sample was included as a negative control. The same blot was also probed for TIRAP and actin to control loading.

  at 1/250, mouse anti-myc9E10 (developed by Bishop, J.M. was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa), mouse anti-HA (Eurogentec, clone 16B12, ref. MMS-101R), rabbit anti-HA (Sigma, ref. H6908), rabbit anti-GFP (Amsbio, ref. TP401), rabbit anti-UBAP1 (Proteintech, ref. 12385-1-AP), mouse anti-His (Sigma, clone HIS-1, ref. H1029), mouse anti-FLAG (Sigma, clone M2, ref. F1804) all at 1/1000 and mouse anti-

20 .

 20 The volume is dependent on the protein concentration of the cell extract. As a guide, 125-150 µg of protein of a cell extract is usually incubated per microgram of bait protein. Alternatively, cell extract samples can be normalized by visualization of transfected proteins to ensure equivalent expression of the prey and the relevant controls (see Note 7). 21. Several controls should be added at this step. Load 400 µL equilibrium buffer without prey protein to analyze the efficiency of the immobilization of the bait protein. As negative controls, load onto the negative column (see Note 12) 200 µL cell lysate containing the prey protein or the negative cell lysate (see Note 7) mixed with 200 µL equilibrium buffer. Additionally, load 200 µL negative cell lysate mixed with 200 µL equilibrium buffer onto the column associated with the bait protein.

Table 2 : Brucella T4SS effector proteins

 2

					Translocation
	Name	Gene	Target	Function	Method	References
	VceA	bab1_1652	Unknown	Inhibits autophagy and induces apoptosis	TEM1	[106]
	VceC	bab1_1058	BiP	Activates UPR	TEM1	[106]
	BPE005	bab1_2005	Unknown	Inhibition of matrix metalloproteinase 9	CyaA	[104]
	BPE043	bab1_1043	Unknown	Unknown	CyaA	[104]
	BPE275	bab1_1275	Unknown	Unknown	CyaA	[104]
	BPE123	bab1_0123	ENO-1	Contributes to intracellular lifestyle. ENO1 recruitment to the BCV	CyaA	[104]
	RicA	bab1_1279	Rab2	Regulation of vesicular traffcking. Rab2 recruitment at the BCV	TEM1	[84]
	BspA	bab1_0678	Unknown	Inhibits the secretory pathways	TEM1 and CyaA	[83]
	BspB	bab1_0712 COG complex	Inhibits the secretory pathway. Biogenesis of rBCV and bacterial proliferation	TEM1 and CyaA	[83]
	BspC	bab1_0847	Unknown	Unknown	TEM1 and CyaA	[83]
	BspE	bab1_1671	Unknown	Unknown	TEM1 and CyaA	[83]
	BspF	bab1_1948	Unknown	Inhibits the secretory pathways	TEM1 and CyaA	[83]
	BtpA/TcpB/Btp1 bab1_0756 TIRAP, Myd88	Inhibits TLR pathways Modulation of microtubule dynamics UPR induction	TEM1 and CyaA	[119]
	BtpB/Btp2	bab1_0279	Myd88	Inhibits TLR pathways	TEM1 and CyaA	[117]
	SepA	bab1_1492	Unknown	Inhibits BCV fusion with the lysosome	3xFLAG	[105]

  The objective of this thesis is to characterize new bacterial effectors identified in Brucella. My work focused on 4 Brucella effectors, BtpA and BtpB, and two new effectors NyxA and NyxB.BtpA and BtpB are two TIR domain-containing proteins known to interfere with TLR-mediated signalling and thereby inhibit the host's innate immune response. Recently, proteins containing TIR domains have been described as having NAD+ hydrolase enzymatic activity in vitro. In collaboration with the team led by Dr. Victor Cid, we aimed to determine if this NAD+ hydrolase activity is retained for BtpA and BtpB. Victor Cid's lab, using over-expression of

4. The different research projects of my thesis work:

these TIR effectors in yeast was able to show that both proteins resulted in NAD depletion in yeast cells. Our team, by measuring the intracellular level of NAD, confirmed these results in epithelial cells expressing BtpA and BtpB but also during Brucella infection. This collaborative work is included in the results, and was published in PloS Pathogens this year, in which I am joint-first author.

Table 3 . Bacterial TIR domain-containg proteins and immune subversion adapted from [169] Protein

 3

		Bacterium	Function	Interaction	References
	BtpA	Brucella spp.	Inhibit TLR2, TLR4 and	TIRAP	[169]
	(Btp1/TcpB)		TLR5-mediated signaling	MyD88	[118]
			Stabilization of	TLR4	[174]
			microtubules	Microtubule	[175]
			Inhibition of dentritic cell	Lipid raft	[120]
			maturation		[176]
			Induces the unfolded		[119] [117]
			protein response (UPR)		[121]
			Facilitate bacterial		[177]
			colonization		[178]
	BtpB	Brucella spp.	Inhibit TLR2, TLR4, TLR5	MyD88	[117]
			and TLR9-mediated
			signaling
			Promote virulence
	TlpA	Salmonella	Inhibit TLR signaling	Not known	[167]
		enterica	responsible for activation
			of NF-κB
			Promotes bacterial
			survival
	TirS	Staphylococcus	Inhibit TLR2, TLR4, TLR5	Not known	[179]
		aureus	and TLR9-mediated		[180]
			signaling
			Promote virulence
	YpTdp	Yersinia pestis	Supresses NF-κB	MyD88	[181]
			activation
			No role in virulence
	PdTlp	Paracoccus	Not known	MyD88	[182]
		denitrificans		TLR4
	TcpC	Escherichia coli	Inhibit TLR2 and TLR4-	MyD88	[118]
			mediated signaling	TLR4	[168]
			Facilitate bacterial		[183]
			colonization		[184]
	PumA	Pseudomonas	Inhibit TLR4 and TLR5-	Myd88	[185]
		aeruginosas PA7	mediated signaling	TIRAP
			Inhibit NF-κB activation	UBAP1
			Promote virulence	Lipid raft

50 .

 50 Rodriguez-Escudero I, Andres-Pons A, Pulido R, Molina M, Cid VJ. Phosphatidylinositol 3-kinasedependent activation of mammalian protein kinase B/Akt in Saccharomyces cerevisiae, an in vivo model for the functional study of Akt mutations. J BiolChem. 2009; 284(20):13373-83. Epub 2009/03/ 25. https://doi.org/10.1074/jbc.M807867200 PMID: 19307184 51. Cid VJ, Shulewitz MJ, McDonald KL, Thorner J. Dynamic localization of the Swe1 regulator Hsl7 during the Saccharomyces cerevisiae cell cycle. Mol Biol Cell. 2001; 12(6):1645-69. Epub 2001/06/16. https:// doi.org/10.1091/mbc.12.6.1645 PMID: 11408575 52. Martin H, Arroyo J, Sanchez M, Molina M, Nombela C. Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C. Mol Gen Genet. 1993; 241(1-2):177-84. Epub 1993/10/01. https://doi.org/10.1007/bf00280215 PMID: 8232202. 53. Sporty JL, Kabir MM, Turteltaub KW, Ognibene T, Lin SJ, Bench G. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae. J Sep Sci. 2008; 31 (18):3202-11. Epub 2008/09/03. https://doi.org/10.1002/jssc.200800238 PMID: 18763242 54. Blasi E, Mathieson BJ, Varesio L, Cleveland JL, Borchert PA, Rapp UR. Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus. Nature. 1985; 318(6047):667-70. https://doi.org/10.1038/318667a0 PMID: 4079980. 2. Brucella effectors target SENP3, inducing subcellular mislocalisation of nucleolar proteins and induction of ribophagy 1. Two newly identified B. abortus effectors, NyxA and NyxB, accumulate in cytosolic and nuclear structures Bacterial effectors often contain eukaryotic-like domains to enable efficient modulation of cellular pathways. Previous work highlighted a subgroup of Brucella candidate effectors containing a carboxyl-terminal CAAX tetrapeptide motif (C corresponds to cysteine, A to aliphatic amino acids and X to any amino acid) [277] . Several bacterial effectors rely on this kind of motif as a lipidation site to facilitate membrane attachment, such as SifA from

	Salmonella

enterica [278] [279]

and AnkB from Legionella pneumophila

[277,280]

. We have recently confirmed that one of these Brucella candidate effectors, BspL, is translocated into host cells during infection although no function has yet been assigned for its CAAX sequence

[281]

. Therefore, we set out to determine if other B. abortus encoded proteins with a potential CAAX box could be translocated into host cells during infection. We relied on the TEM1 ßlactamase reporter, widely used to assess Brucella effectors' translocation, to test two CAAXcontaining proteins encoded by the genes BAB1_0296 (BAB_RS17335) and BAB1_0466

B Samples 18_0106-0107 -Gel from 16/02/2018 Ref. PSF Ref. Labo LCMSMS Analysis (Q Exactive HF) Database Enzyme Results File PD 2.1 18_0107 Checked

												# Peptides Sequest HT
	TRUE	Master Protei NyxA	ALOUCHE_1802	86,6	79,3	14	99	7	1	15,2	150,4	14
	TRUE	Master Protei HisNyxB	ALOUCHE_1802	26,2	38,9	9	60	2	1	18,8	87,0	9

NyxA

Master Accession 180302-0107-01.raw Description Sum PEP Score Coverage # Peptides uniprot-brucella-abortus_1803 + séquences HisBnpB et BnpA # PSMs nique PeptideProtein Groups MW [kDa] Chymotrypsin Score Sequest HT 180302-0106-01.pdResult

Table 4 . Eukaryotic proteins interacting with NyxA identified with the yeast two-hybrid (Y2H) screen.

 4

Table 1 . Antibodies used in this study.

 1

	Antibody	Species	Source	Reference	Dilution
	SENP3	Rabbit	Cell Signalling	5591	1/400
	NVL	Rabbit	Proteintech	16970-1-AP	1/25
	RPL5	Rabbit	X		1/100
	PES1	Rabbit	ATLAS antibodies	HPA040210	1/100
	NPM1	Rabbit	Abcam	Ab37659	1/400
	Nucleolin	Mouse	Invitrogen	39-6400	1/100
	HA	Rat			1/100
	Flag	Mouse	Sigma	F1804 (clone M2)	1/2000
	Myc	Mouse	DSHB	(Clone E10)	1/1000
	NUFIP1		Proteintech	12515-1-AP	1/50
	LAMP1	Mouse	DSHB	(Clone H4A3)	1/200
	FK2	Mouse	Enzo	BML-PW8810	1/1000
	PML	Rabbit	Abcam	Ab53773	1/500
			Western blot
	Actin	Mouse	Sigma	A4700	1/1000
	His	Mouse	Sigma	H1029	1/3000
	V5	Mouse	Invitrogen	R960-25	1/1000
	SENP3	Rabbit	Cell Signalling	5591	1/1000
	NPM1	Rabbit	Abcam	Ab37659	1/400
	Histone 3	Rabbit	Abcam	Ab8895	1/500
	PML	Rabbit	Abcam	Ab53773	1/500
			Secondary antibodies
	Donkey anti-mouse	Invitrogen	A-21202/A-	1/1000
	AlexaFluor488/555/647		31570/A-31571
	Donkey anti-rabbit	Invitrogen	A-21206/A-	1/1000
	AlexaFluor488/555/647		31572/A-31573
	Donkey anti-rat		Invitrogen	A-21208/A-	1/1000
	AlexaFluor488/555/647		21434/A-21472

Table 2 . Primers used in this study.

 2

	Purpose		Sequence 5'-3'
	pNPTS138-	Fw1 GATAATGACGCTAGCTTTCA
	NyxA	Rev1 GGCCACCCGACAAGCAGTTTGGAGGTTTACCTTTTGTTGA
		Fw2 TCAACAAAAGGTAAACCTCCAAACTGCTTGTCGGGTGGCC
		Rev2 GGGTGTCCTTGAAACATCCA
	pNPTS138-	Fw1 TTGGATCAATCCGGCGTGTG
	NyxB	Rev1 GGGCTTCAACTTCTTTAACCGGCTATTCTTCCTGTCAATT
		Fw2 AATTGACAGGAAGAATAGCCGGTTAAAGAAGTTGAAGCCC
		Rev2 CGTAAACGCTTCGGCAGGGA
	pFlagTEM1-	fw	TAAGCATTGGTCTAGAATGAACGCTCACACAAACATAA
	NyxA	rev	ACTGCAGTTATCTAGATCAAAGCTCCAAGCATCTAATT
	pFlagTEM1-	fw	GAGATAGGTGCCTCACTGATTAAGCATTGGTCTAGAATGAACACGCAAGCAACAATA
	NyxB	rev	GTGTGCTGGAATTCGCCCTTACTGCAGTTATCTAGATCAAGGCATCTCGATAAG
	pFlagTEM1-	fw	TAAGCATTGGTCTAGAATGAAAATGTGGACCCTTGC
	BAB1_0466	rev	ACTGCAGTTATCTAGATCACTGTTCTACGCAGCTTA
	pTn7-nyxA	fw rev	GTGAAATCAATCAACAAAAGGTAAACCTCCatgaacgctcacacaaacataag GGAATTCCTGCAGCCCGGGGGATCCACTAGtcaaagctccaagcatctaatttc
	pTn7-nyxB	fw rev	ATTTAACCGAAATTGACAGGAAGAATAGCCatgaacacgcaagcaacaatag GGAATTCCTGCAGCCCGGGGGATCCACTAG tcaaggcatctcgataaggc
	pBBRMCS2	fw	aaaaaaGAGCTCaaggagatatacatatgTACC
	4HA-nyxA	rev	aaaaaaACTAGTtcaaagctccaagcatctaatttc
	pBBRMCS2 3Flag-nyxA	fw rev	TATTCCCGGGGGATCCATGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATT CTACGAACGCTCACACAAACATAAG TATAGGGCGAATTGGAGCTCAAGCTCCAAGCATCTAATTT
	pBBRMCS2	fw	aaaaaaGAGCTCaaggagatatacatatgTACC
	4HA-nyxB	rev	aaaaaaACTAGTtcaaggcatctcgataaggc
	pDONOR-	fw	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAACGCTCACACAAACATAAG
	NyxA	rev	GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAAGCTCCAAGCATCTAATTTC
	pDONOR-	fw	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAACACGCAAGCAACAATAGATA
	NyxB	rev	GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAGGCATCTCGATAAGGCGGATT
	Pet151His-	fw	CACCATGAACGCTCACACAAAC
	NyxA	rev	TCAAAGCTCCAAGCATCT
	Pet151His-	fw	CACCATGAACACGCAAGCAAC
	NyxB	rev	CATTATGCTCCCCTGTTGT
	NyxA MAG	fw rev	aaaaaaGAGCTCTAAGTGTCTGCCATAGCCGACG aaaaaGGATCCtcaaagctccaagcatctaatttc
	NyxA Y62R	fw rev	CGATTGGcgaCCTGCCGCCTATGATG CGGCAGGtcgCCAATCGTAGCAGTCGAAG
	NyxA D76R	fw rev	CCATGAAAcgaCGGGAACTGATCCAATACG TTCCCGtcgTTTCATGGCGTTGCCTTC
		fw	CGACGGagaCTGATCCAATACGAAGAGTGGTG
	Nyx E78R	rev	GGATCAGtctCCGtcgTTTCATGGCG
	NyxA MAG	fw rv	aaaaaaGAGCTCTTGGATCAATCCGGCGTGTGC aaaaaGGATCCtcaaggcatctcgataaggc
	SENP3 7-159 Fw	CACCATGGCCGGCACCGGTAGCTGGGGTCCGGAACC
		rv	TTTGGATCCTTATTTGCTATACAGCAGCATACGAAATGC

  Plasmid containing the gene of interest fused to a specific tag (obtained from a EndoFree maxipreparation). 4. Transfection reagent. 5. Phosphate buffered saline (PBS): Prepare a 10× solution with bidistilled water (18.2 MΩ cm) containing 10.6 mM KH2PO4, 30 mM Na2HPO4, 2H2O, and 1.54 M NaCl, and sterilize with a 0.2 µm filter. The 1× solution obtained following dilution with bidistilled water will have a pH of around 7.4. 6. Radioimmunoprecipitation assay (RIPA) buffer: Ready-to-use solution containing 150 mM NaCl, 1.0% IGEPAL ® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0. 7. Antiprotease cocktail: Mix 1% (v/v) of protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), phosphatase inhibitor cocktail 3 (Sigma-Aldrich), and phenylmethylsulfonyl fluoride (PMSF). 1. 1 M Tris-HCl, pH 7.5 stock solution. Weigh 121.1 g Tris base and transfer to a 1 L graduated cylinder. Add water to 800 mL, mix, adjust pH with HCl, and make up to 1 L with water. Store at room temperature (see Note 1). 2. 5 M NaCl stock solution. Weigh 292.2 g NaCl and transfer to a 1 L graduated cylinder. Add water to 800 mL, stir, and adjust volume to 1 L with water (see Note 1). 3. Equilibrium buffer (see Note 2): 20 mM Tris-HCl, pH 7.5, 250 mM NaCl. Mix 1 mL 1 M Tris-HCl, pH 7.5 stock solution with 2.5 mL 5 M NaCl stock solution in a 50 mL centrifuge tube, and add water to a volume of 50 mL. Keep at 4 °C (see Note 3). 4. Elution buffer (see Note 2): 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 500 mM imidazole. Weigh 1.7 g imidazole in 50 mL solution of equilibrium buffer. Keep at 4 °C (see Note 3). 5. Purified His-tagged protein (bait). 6. Ni-NTA agarose beads: 6% beaded agarose (cross-linked), precharged with Ni 2+ (Protino ® Ni-NTA Agarose, Macherey Nagel, or equivalent). Store at 4 °C (see Note 4). 7. 0.8 mL empty columns for gravity flow (Pierce™ Centrifuge Columns, Thermo Fisher Scientific, or equivalent). 8. Refrigerated microcentrifuge. 1. Resolving gel: 1.5 M Tris-HCl, pH 8.8. Weigh 90.8 g, transfer to 500 mL graduated cylinder, and add 300 mL water. Adjust pH with HCl and fill with water to 500 mL. Store at room temperature. 2. Stacking gel buffer: 0.5 M Tris-HCl, pH 6.8. Weigh 30.275 g, transfer to 500 mL graduated cylinder, and add 300 mL water. Adjust pH with HCl and fill with water to 500 mL. Store at room temperature. 3. 30% acrylamide/Bis solution (37.5:1 acrylamide:Bis). Store at 4 °C. 4. Ammonium persulfate (APS): 20% solution in water. Store at -20 °C (see Note 5). 5. N,N,N',N'-tétraméthyléthylènediamine (TEMED). Store at room temperature. 6. SDS-PAGE running buffer: 25 mM Tris-HCl, 192 mM glycine, 0.1% SDS. Prepare 10× running buffer solution: Weigh 30 g Tris base, 144 g glycine, and 10 g SDS and add distilled water to 1 L. Store at room temperature. Prepare fresh 1× solution before gel electrophoresis. 7. Laemmli lysis buffer [6], 4× concentrate: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% p-mercaptoethanol. Store at -20 °C (see Note 6). 8. Protein ladder.

	3.
	2.2 Pull-
	Down Assays
	2.3 Sodium Dodecyl
	Sulfate (SDS)
	Polyacrylamide Gel
	Components

  4. Centrifuge 5 min at 80 × g at 4 °C. 5. Resuspend cells with 200 µL RIPA buffer supplemented with antiprotease cocktail. 6. Incubate on ice 20 min and mix gently every 5 min with a P200 micropipette. 7. Stock prepared cells at -80 °C (see Note 8). 8. Right before pull-down experiment, thaw prepared cell extract. Centrifuge at 17,000 × g at 4 °C for 20 min. Use the supernatant as prey by following step 9 in Subheading 3.2 (see Note 9). . Equilibrate column by adding 400 µL equilibrium buffer supplemented with 20 mM imidazole. 4. Centrifuge column for 1 min at 1000 × g at 4 °C. Discard flow-through. 5. Load 400 µL incubated bait and prey proteins onto column. Incubate 10 min on ice without agitation (see Note 18). 6. Centrifuge column for 1 min at 1000 × g at 4 °C. Keep flowthrough at 4 °C for analysis. 7. Wash by adding to column 400 µL equilibrium buffer supplemented with 20 mM imidazole. 8. Centrifuge column for 1 min at 1000 × g at 4 °C. Save first washing at 4 °C for analysis. 9. Repeat washing steps 7 and 8 four times and keep last washing fraction at 4 °C for analysis. 10. Add 200 µL elution buffer to column and incubate on ice 10 min. 11. Centrifuge column for 1 min at 1000 × g at 4 °C. Keep eluted fraction at 4 °C for analysis.

1. Transfer 120 µL Ni-NTA agarose beads to gravity flow column (see Note 12).

2. Centrifuge column for 1 min at 1000 × g at 4 °C. Discard flow-through.

3

PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007979April 16, 2020

PLOS Pathogens | DOI:10.1371/journal.ppat.1006092December 29, 2016

TheEMBO Journal Vol 36 | No 13 | 2017 ª 2017 The Authors

ª 2017 The Authors The EMBO Journal Vol 36 | No 13 | 2017

Acknowledgments

We wish to acknowledge the Genomics and Mass Spectrometry Units at Universidad Complutense de Madrid (UCM) for sequencing and NAD + analyses, respectively. We would like to thank Thomas Henry (CIRI, Lyon) for proving us with iBMDM and Diego Comerci (CONI-CET, Argentina) for helping us with the complementation constructs. We also thank Lucia Sastre for technical assistance in molecular cloning and yeast growth assays and members of U3 Research Lab for help and discussion.

Acknowledgments

We gratefully acknowledge Vale´rie Vogel and David Hernandez for help concerning bioinformatics analysis, and Myriam Girard, Christophe Ginevra and He ´lène Meunier for technical assistance. We thank Christopher Montgomery and Lloyd Miller for mouse model consultation. We also thank Jacqueline Marvel for helpful discussions. We are grateful to Laurent Genestier for helpful discussions and for providing MyD88-deficient mice and to the staff members at PBES, with a particular thanks to Nadine Aguilera for their support in the animal facility.

Acknowledgements

This work was funded by the FINOVI foundation under a Young Researcher Starting Grant and the Cystic Fibrosis French Foundation Vaincre la Mucovicidose (VLM), grant RF20130500897. SS and SB are supported by INSERM and CNRS staff scientist contracts, respectively. SG and JBL are funded by the Région Rhônes-Alpes ARC1 Santé fellowships. PI and AL by the VLM and FINOVI grants. TW is supported by a Wellcome Trust PhD fellowship. We thank the following people: L. Plantevin for programming of ImageJ plugin; V. Gueguen-Chaignon and the Protein Science Facility (SFR Biosciences, France) for protein purification and plasma resonance experiments; R. Voulhoux (CNRS UMR7255, Aix-Marseille University, France) for anti-EF-Tu and LasB antibodies, PA7 strain and vectors pKNG208; G. Ball (CNRS UMR7255, Aix-Marseille University, France) for advice on genetics of PA7; S. Lory (Harvard Medical School, USA) for anti-PilQ antibody; pCMV-HA-MyD88 was a gift from B. Beutler (UT Southwestern Medical Center, USA; Addgene plasmid # 12287), FLAG-TLR5 from R. Medzhitov (Yale University, USA; Addgene plasmid # 13088), TIRAP-GFP and GFP-MyD88 from J. Kagan (Harvard Medical School, USA); HA-TIRAP from L. O'Neil (Trinity College Dublin, Ireland); Myc-TIRAP from A. Weber; L. Alexopolou (CIML, France) FLAG-TLR2 and FLAG-TLR4. We also thank the PLATIM of the SFR Biosciences for help with microscopy, T. Henry (CIRI, Lyon) for discussion and J. Kagan (Harvard Medical School, USA) for discussion and critical reading of the manuscript.

This work was funded by the FINOVI foundation under a Young Researcher Starting Grant, the Cystic Fibrosis French Foundation Vaincre la Mucovicidose grant RF20130500897 and the ANR (grant n˚ANR-15-CE15-0011) to SS, and by grants BIO2016-75030-P from Ministerio de Econom´ıa y Competitividad (Spain) and S2017/ BMD-3691 (InGEMICS-CM) from Comunidad de Madrid and European Structural and Investment Funds to VJC and MM. SS is supported by an INSERM staff scientist contract. J.M.C-S is supported by a predoctoral contract from UCM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding: This work was supported by the LABEX ECOFECT (ANR-11-LABX-0048) of Universite´ de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR) and a FINOVI Young Researcher Grant and by the Swiss Here, we investigate TirS, a National Science Foundation grant 31003A_153474. SP was supported by the Innovative Medicines Initiative Joint Undertaking under the Combatting Bacterial Resistance in Europe (COMBACTE) grant agreement no. 115523. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ENA database project study accession number PRJEB12840; sample accessions ERS1070204 to ERS1070207 and ERS1434451, ERS1434452.

Chapter II: Results

PLOS PATHOGENS

Brucella TIR proteins deplete host cell NAD + phalloidin for labelling actin (cyan). (B) GFP-BtpB (green) can be detected at intercellular contacts (arrow and zoomed image). Cells were labeled with an anti-tubulin antibody (red). (C) HeLa cells were transfected with GFP-BtpB-TIR (green) and then labelled for tubulin (red) or (D) vimentin (red). (E) Representative image of cells labelled with anti-tubulin antibody (red) expressing aggregates of GFP-BtpB E234A (green). Scale bars correspond to 5 µm. For both effectors, punctate and filament-like structures were observed (Figure 23A). Equivalent NyxA structures were observed with an N-terminal 3Flag (Figure 23B), suggesting these are not an artefact due to the 4HA tag.

NyxA and NyxB interact with each other and target the same cellular compartments

Imaging of translocated NyxA and NyxB suggested that these effectors may have an identical subcellular localisation. To investigate this possibility, we ectopically expressed NyxA and NyxB with different tags. We found that HA-tagged NyxA and NyxB predominantly accumulated in

Bacterial expression vectors

NyxA and NyxB were amplified by PCR and inserted into the pET151D topo vector following the manufacturer's procedure (Invitrogen) to obtain His-NyxA and His-NyxB. An additional V5

tag is also present. His-NyxA MAG and His-NyxB MAG were obtained by site-directed mutagenesis.

For expression and purification of SENP37-159, a vector with E. coli codon-optimized SENP3 was obtained from Thermofisher and used as a template. All the primers used are listed in Supplementary Table 2.

Immunofluorescence microscopy

At the indicated time points, coverslips were washed twice with PBS, fixed with PFA 3.2%

(Electron Microscopy Sciences) for 20 minutes and then washed again 4 times with PBS. Best NVL staining was achieved with AntigenFix (MicromMicrotech France). For SENP3, NVL, RPL5, PES1 and NUFIP1 immunostaining, permeabilization was carried out with a solution of PBS containing 0.3% triton for 30 minutes followed by blocking also for 30 minutes in a solution of PBS containing 2% bovine serum albumin (BSA), 10% horse serum, 0.3% triton. For HA and Flag to detect translocated effectors blocking and antibody solutions did not contain Triton (only the permeabilization step). For NPM1 staining Permeabilization was done with Tween 0.05% followed by antibody diluted in 2% BSA, 10% horse serum and 0.3M glycine. Coverslips were then incubated at 4 °C overnight with primary antibody diluted in the blocking solution.

Subsequently, the coverslips were washed twice in PBS and incubated for 2h with secondary antibodies. When indicated DAPI was also included in the secondary antibody mix (1/1000 suggesting that NyxA exhibits a non-functional CAAX motif. Indeed, preliminary data from the lab using HeLa cells were transfected with a plasmid encoding myc-NyxA or myc-NyxAΔCLEL, showed no difference in localization between these proteins suggesting that this CAAX motif may not be functional (Figure 50). Subsequently nyxB was identified in the Brucella genome encoding for a protein showing 82% identity with the sequence of NyxA. It is somewhat surprising that Brucella produces two highly similar effectors, yet this must be advantageous for the bacterium, otherwise during evolution Brucella would have lost one of the two genes.

The first step of our study was to confirm that these two potential effectors were translocated in the host cell during infection. For this, a reporter system, TEM1 ß-lactamase reporter, was used. We found that the translocation of NyxA is dependent on the T4SS present in Brucella but this does not seem to be the case for NyxB. This observation is rather surprising and raises many questions. The position of the reporter tag on the N-terminus of the effectors, may interfere with the translocation signal and perturb its association with the T4SS. VirB operon could be deleted. Consistent with our results, several Brucella effectors appear to be translocated independently of T4SS (for example, BspD, G, H, I, J, K) [START_REF] Myeni | Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins[END_REF]. It therefore seems necessary to better characterize the molecular mechanisms involved in the translocation of effector protein in the host cell by the Brucella T4SS VirB to determine if an alternative secretion system is contributing. Perhaps NyxA and NyxB, highly similar but with clear translocation mechanisms, could provide an interesting tool to study these questions.

ANNEXES Author Summary

Pathogenic microbes have evolved elaborate strategies to manipulate host defenses to establish and spread in the host population. One such mechanism involves disruption of the immune signaling cascade orchestrated by the Toll-like receptors (TLRs), which sense

Conflict of interest

The authors declare that they have no conflict of interest.