This thesis is missing page 53, no other copies have this page. -Digitization Centre / Bacterial adaptation to changing conditions and to the host environment requires coordinated changes in gene expression that permit more efficient utilization of metabolites and increased survival. An important form of gene control is through the use of alternative sigma factors that direct RNA polymerase to recognize a distinct group of genes. One such sigma factor is RpoS, which is widely present in Proteobacteria including many serious human pathogens. As a key stress response regulator, RpoS plays an important role in adaptation, but its effect on virulence varies in different species. RpoS contributes to virulence through either enhancing survival against host defense systems or directly regulating expression of virulence factors in some pathogens, while RpoS is dispensable, or even inhibitory, to virulence in others. The primary objective of this study is to understand the mechanism of RpoS control in gene expression and pathogenesis of Escherichia coli. This thesis first describes the characterization of RpoS regulon in laboratory and pathogenic E. coli strains by transcriptome profiling analysis. Comparison of RpoS regulons identifies a core set of RpoS-controlled genes as well as strain-specific groups of genes, including many implicated in virulence. The contribution of RpoS to enteropathogenesis in vivo was tested using a Citrobacter rodentium (CR)mouse infection model that is commonly used to simulate E. coli infection in human intestine. Mutations in rpoS result in reduced colonization and delay in mortality, indicating RpoS is important for full virulence. Clinical and natural E. coli isolates exhibit variable abilities in stress resistance and virulence, which is partly attributable to attenuating polymorphisms of rpoS commonly found in E. coli populations. A possible mechanism responsible for the occurrence of rpoS polymorphisms in pathogenic E. coli is addressed. Using a group of representative enterohemorrhagic E. coli strains, we report that growth-enhanced mutants can be selected during growth on succinate and other poor carbon sources under both aerobic and anaerobic conditions. The majority of these mutants carry nonsense or missense mutations in rpoS. Phenotypic microarray analysis reveals that rpoS mutations result in increased utilization of 92 nitrogen and 8 carbon sources. Therefore, the occurrence of rpoS polymorphisms may increase the fitness of the population as a whole for better nutrient scavenging. In conclusion, RpoS may be viewed as a transient regulator that orchestrates the temporal expression of a large regulon for better adaptation under specific conditions including natural and host environments. Under conditions not requiring RpoS, its functions can be turned off through decreasing expression, rapid proteolysis, inhibition of RpoS activity, or selection of attenuating mutations. The final part of this thesis reviews the distinct and niche-dependent involvement of RpoS in virulence of many rpoS-bearing pathogens. - / Thesis / Doctor of Philosophy (PhD)

Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250 / Ph. D.

Pseudomonas aeruginosa est un pathogène opportuniste responsable des infections pulmonaires chroniques chez les patients atteints de fibrose kystique (FK). L’émergence de nouvelles souches épidémiques hypervirulentes, multi-résistantes aux antibiotiques et possédant une grande capacité de transmission, telles que LESB58, entraîne de grandes difficultés de traitement et une augmentation de la mortalité chez ces patients. Le séquençage complet de LESB58 a révélé un génome à 90% hautement conservé additionné de 455 gènes regroupés sous six prophages (PPs) et cinq îlots génomiques (GIs). Cette étude a pour objectif de déterminer l’implication de ces régions supplémentaires dans la pathogénie de LESB58. Une mutagenèse à étiquette signature (STM) suivie de plusieurs criblages ont permis de sélectionner 162 mutants dont 11 portant une insertion dans un GI ou PP. Des analyses subséquentes ont été réalisées sur ces mutants afin d’évaluer leur niveau de virulence in vivo dans un modèle d’infection pulmonaire chronique chez le rat. Des analyses de génomique ont été effectuées sur deux de ces mutants afin de mieux comprendre leur incapacité à établir l’infection dans le modèle d’infection in vivo. / Pseudomonas aeruginosa is an opportunistic pathogen and causes chronic pulmonary infection to cystic fibrosis (CF) patients. The hypervirulent epidemic strain LESB58 is associated with high transmissibility and is highly resistant to antibiotics causing increased morbidity and mortality. Whole genome sequencing of LESB58 revealed a 90% highly conserved core genome and 455 additional genes grouped in five genomics islands (GIs) and six prophages (PPs). The aim of this study was to determine the implication of the accessory genome in LESB58 virulence. We performed a signature tagged mutagenesis (STM) followed by an in vivo screening of the mutants. From 162 STM mutants defective for in vivo maintenance, we selected 11 harbouring an insertion in GI or PP for further virulence analysis. Two of these mutants were used for genomics analysis in order to better understand their incapacity for in vivo maintenance in the rat model of chronic lung infection.

Plants are surrounded by various threats from the environment such as pathogens, abiotic stresses, and animal attacks. Nutrient content and distribution are essential for plant growth and development as well as plant immunity. Pathogens extract nutrients from host plants to benefit their own growth and reproduction. Sulfate, amino acids, and phosphate are indispensable elements for plant growth, plant nutrition, and plant resistance/susceptibility to disease. However, the role of these nutrients in plant-oomycete interactions is an unexplored area. We developed a hydroponic system to precisely control the nutrients applied to plants. We used Arabidopsis thaliana and Nicotiana benthamiana (N. b) as model plants. Hyaloperonospora arabidopsidis as well as two Phytophthora species, Phytophothora capsici (P. cap) and Phytophothora nicotianae (P. nic) were used as model oomycete pathogens. Hpa is an obligate biotrophic pathogen that obtains nutrients directly from the host plant without causing cell death, while P. cap and P. nic are hemibiotrophic pathogens that display a biotrophic phase followed by a necrotrophic phase where they feed on dead cells. Genomic evidence suggests that these pathogens might obtain nutrients including sulfur in different forms from the host (organic and inorganic respectively). We have optimized the hydroponic system and used Taqman PCR assays and sporangiophore counts to assay the influence of sulfur nutrients on Hpa and P. cap infections. We found that (1) sulfur transporter and metabolism genes play essential roles in plant-oomycete interactions; (2) sulfur is critical components for HR responses against Hpa; (3) low sulfur induces pathogenesis related genes as a systemic acquired response. RNA-seq analysis on Phytophthora-infected Arabidopsis suggested that sulfur transport, assimilation, and metabolism play an important role in plant-oomycete interactions. A second project used RNA-seq analysis on P. nic infected N. b, to identify potential nutrition-related-plant genes that are necessary for full pathogen virulence. RNAi knockdowns of N. b AAP6 (amino acid permease 6) and PHT4 (phosphate transporter 4) genes showed an inhibition of oomycete colonization. These experiments together advance the study on the interplay between nutrient assimilation/metabolism in host plants and oomycete infection which will provide insight into the mechanisms how pathogens intercept nutrients from host. In the long-term, this research could reveal new traits applicable for disease resistance to promote crop and food production. / Doctor of Philosophy / Plants are surrounded by diverse threats from the environment such as pathogens, abiotic stresses, and animal attacks. Oomycetes are the most destructive group of pathogens, triggering severe food security issues. Phytophthora is an oomycete genus causing serious economic loss. Traditional disease control managements including pesticides, crop rotation and culture practices, are not time- or financially- efficient due to the difficulty in managing oomycete spread and oomycete resistance to chemicals. Thus, new plant genes for resistance to oomycete diseases would have a major impact. Plant nutrients are critically important for plant fitness in every aspect of plant growth and plant immunity. Cellular regulatory networks for sulfur, amino acids, and phosphate assimilation and metabolism networks connect to every aspect of plant activity such as functioning enzymes, formation of chlorophyll, synthesis of proteins, and plant immunity. These nutrients are part of the plant defense system but also can be beneficial nutrients fed to the invading pathogens. Studying how nutrients are involved in the responses to oomycete invasions will provide information to introduce resistance strategies into crops. We utilized oomycete pathogens with different lifestyles to study the interactions and found that some sulfate transporter genes, an amino acid transporter and a phosphate transporter might be manipulated by oomycete to obtain nutrients. Sufficient nutrition is a critical factor for successfully triggering plant immunity but also could be reprogrammed by pathogens for successful infection and development. Our studies gave useful information to understand which plant nutrient genes are important during plant–oomycete interactions. These findings could be useful in identifying or engineering new plant genes to control plant diseases.

Protein complexes mediate a diverse range of behavior in prokaryotic cells, yet the exact molecular mechanisms explaining how many of these complexes assemble and function remain unknown. This work focuses on understanding the molecular mechanisms of two different protein assemblies responsible for regulating virulence in the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa utilizes type IV pili (T4P) to adhere to, and move along, surfaces. Assembly of T4P is powered by a dedicated cytoplasmic ATPase, PilB. The structural study of PilB from a related system (chapter 2) resulted in the formulation of the first model describing the mechanism of force generation resulting from ATP hydrolysis, which explains how T4P are assembled. Chapter 3 focuses on the RetS/GacS interaction, which is responsible for globally regulating virulence in P. aeruginosa. A comprehensive structural study reveals a dynamics of a novel regulatory interaction and the discovery of a potentially universal transmembrane signaling mechanism. / Doctor of Philosophy / Bacteria have threatened human health since the beginning of recorded history. With the development of antibiotics in the early twentieth century, the threat posed by bacterial infection was greatly lessened. However, decades of antibiotic mismanagement has led to the evolution of bacteria which are no longer vulnerable to these antibiotics. In order to combat this rising threat of resistant bacteria, we require a deeper understanding of how bacteria function and cause disease. Proteins play a crucial role in the diseases caused by bacteria, either by directly damaging host cells or regulating the expression of these damaging factors. By increasing our knowledge of the roles played by protein during bacterial infections, it will be possible to create new antibiotics while minimizing the risk of resistance. The work presented here grants a deeper understanding into how proteins work together to allow bacteria to survive inside the human body.

Blastocystis spp. est un Straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce parasite est associé à des troubles gastro‐intestinaux aspécifiques, et semble impliqué dans des désordres fonctionnels tels que le syndrome de l’intestin irritable (IBS). Ce travail de thèse s’appuie sur le séquençage du génome de Blastocystis sp. ST7 réalisé en collaboration avec le Génoscope d’Evry, l’Université Nationale de Singapour, l’Institut Pasteur de Lille et l’Université de Provence. Ce génome est constitué d’un génome nucléaire de 18,8 Mpb pour 6020 gènes, et d’un génome mitochondrial de 29 kpb localisé dans des organites apparentés aux mitochondries. L’analyse de ce génome apporte des informations au niveau de l’évolution de ce microorganisme, de son adaptation à l’environnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce génome ont montré que Blastocystis sp. ST7 possède plusieurs gènes codant des protéines pouvant agir à l’interface entre l’hôte et le parasite et connues chez d’autres protozoaires pour être impliquées dans des phénomènes de pathogénie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protéases. D’autre part, des activités protéolytiques ont été mises en évidence expérimentalement dans les surnageants de culture du parasite. Deux protéases à cystéines (une cathepsine B et une légumaïne) pouvant être impliquées dans la physiopathologie du parasite, ont été identifiées et caractérisées dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intéressantes à explorer pour évaluer l’impact de ce parasite en santé humaine. / Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the Génoscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria‐like genome of 29 kpb localised in the mitochondrion‐like organelles. The analysis of this genome brings information about the evolution of this micro‐organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite‐host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.

Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence / Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion

Introduction : En 2011, un arbovirus émergent appelé virus Schmallenberg (SBV) et appartenant à la famille des Bunyaviridae a été identifié en Allemagne et s’est répandu en Europe. Le SBV infecte les ruminants domestiques et sauvages. Chez l’adulte, la virémie est transitoire et l’infection est souvent inapparente. En revanche, chez les femelles gestantes, le SBV peut franchir la barrière transplacentaire et infecter le fœtus, pouvant provoquer des avortements et des malformations du système nerveux central. Parmi les protéines virales synthétisées par le SBV, la protéine non-structurale NSs est un facteur de virulence majeur. Elle entraîne notamment la dégradation de sa sous-unité Rpb1 de l’ARN polymérase II pour inhiber la transcription cellulaire. Ce travail a pour but d’étudier les propriétés biochimiques et fonctionnelles de NSs et d’identifier les déterminants moléculaires régissant ses principales activités.Méthodes et résultats: L’analyse in silico de la séquence peptidique de NSs réalisée à l’aide d’algorithmes de prédiction a permis de designer plusieurs de mutants de délétion de la protéine. L’observation de la localisation cellulaire des mutants dans plusieurs modèles humains et ovins confirme la prédiction d’une distribution principalement nucléaire pour NSs. De façon intéressante, une séquence interne à la protéine (33-51) sert de motif d’adressage spécifique au niveau des nucléoles (NoLS) et nous avons pu démontrer la co-localisation de NSs avec plusieurs protéines nucléolaires. De plus, l’infection de cellules humaines et ovines par le SBV entraîne la translocation de protéines nucléolaires (B23 et fibrillarine) vers le nucléoplasme, témoignant d’un stress nucléolaire viro-induit. Pour évaluer l’impact de la localisation nucléolaire de NSs sur ce phénomène, un virus recombinant dont NSs a été délétée de son motif d’adressage nucléolaire (SBVΔNoLS) a été produit par génétique inverse. Le SBVΔNoLS n’induit plus de redistribution de B23 confirmant le rôle de NSs dans l’induction d’un stress nucléolaire au cours de l’infection. Ces résultats ont été confirmés dans des cellules souches neurales humaines, qui constituent un modèle pertinent par rapport aux lésions provoquées par le SBV dans le système nerveux.En parallèle de ce travail, nous avons recherché des partenaires cellulaires de NSs par la méthode du double-hybride en levures. Huit partenaires cellulaires de NSs ont été découverts, dont la chaîne légère de la dynéine de type 1 (Tctex-1) et la Major Vault Protein (MVP), qui sont toutes les deux impliquées dans le transport de protéines grâce à leur association aux microtubules. Une des hypothèses avancées est que ces protéines pourraient servir de cargos pour promouvoir le transport nucléo-cytoplasmique de NSs.Conclusions et perspectives : Ce travail de thèse a permis de démontrer que la protéine NSs du SBV est localisée principalement dans le noyau cellulaire et dans les nucléoles, grâce à une séquence d’adressage spécifique. L’infection virale induit un stress nucléolaire dépendant de NSs, qui a pu être reproduit dans un modèle de cellules souches neurales humaines. Les perturbations nucléolaires induites par NSs pourraient contribuer au blocage de la transcription cellulaire observé au cours de l’infection et, de manière subséquente, moduler la réponse antivirale de la cellule et/ou induire la mort cellulaire en lien avec la pathogenèse virale. Ainsi, ces perturbations des nucléoles pourraient être à l’origine d’une dégénérescence des neurones et des anomalies développementales observées chez les fœtus infectés. Au niveau moléculaire, nous souhaitons préciser l’implication de la protéine nucléolaire B23, relocalisée vers le nucléoplasme en cours d’infection, et/ou d’autres composants du nucléole dans l’initiation de ce processus. Enfin, l’hypothèse d’un transport rétrograde actif de NSs du cytoplasme vers le noyau médié par son interaction avec MVP ou Tctex1 est en cours d’investigation. / Introduction: In 2011, an emerging arbovirus named Schmallenberg virus (SBV), and belonging to the Bunyaviridae family, was discovered in Germany. Then, SBV has rapidly spread to Europe infecting wild and domestic ruminants. Adult infection is basically mild and associated with a short viremia (2-5 days). However, in case of pregnant females’ infection, SBV has the ability to cross the placental barrier to infect the foetuses, which can lead to stillbirth and central nervous system developmental abnormalities (arthrogyposis, hydranencephaly). Among bunyavirus-encoded proteins, the non-structural protein NSs has been shown to be an important virulence factor. Indeed, it is able to degrade the Rpb1 subunit of RNA polymerase II, leading to the inhibition of cellular transcription. The work of my thesis aimed to study biochemical and functional properties of NSs and to identify the molecular patterns ruling its main activities.Methods and results: An in silico amino acids sequence analysis was used to predict some common features of NSs and to help the design of several NSs mutants. As predicted by several algorithms, NSs and its mutants are mainly localised to cell nucleus in different cell types (from human and ovine origin). Interestingly, we highlighted an internal sequence (residues 33 to 51) containing a nucleolar localisation signal (NoLS), and have shown that NSs co-localises with several nucleolar proteins. Moreover, infections of human and ovine cell lines with SBV lead to re-localisation of nucleolar proteins to nucleoplasm (B23 and fibrillarin), demonstrating a viral-induced nucleolar stress. To assess the role of the NSs nucleolar localisation in this phenomenon, a recombinant virus, with a mutated version of NSs devoid of its NolS motif (SBVΔNoLS), was constructed by reverse genetic. Infection with SBVΔNoLS does not induce nucleolar stress, suggesting that the nucleolar stress induced by SBV occurs only if NSs is addressed to the nucleolus. Moreover, these results have been confirmed in human neural stem cells, which coud be a more relevant cellular model to mimic SBV infection in foetuses.Another way to study NSs functions was to identify its cellular partners by means of a yeast-two hybrid screen and using NSs as bait. Eight putative interactors of NSs have been discovered, including the dynein light chain type 1 (Tctex-1) and the Major Vault protein (MVP). These proteins are involved in cellular protein transport, notably by their associations with the microtubules network. Thus, NSs might interact with Tctex-1 and MVP to favour its shuttling from cell cytoplasm to the nucleus.Conclusions and perspectives: Altogether, these data indicate that SBV-NSs protein is mainly localised into cell nucleus and nucleolus, by means of its internal NoLS contained in the 33-51 NSs domain. SBV infection induces a nucleolar stress, particularly in human neural stem cells. NSs-induced nucleolar disruption could promote NSs inhibitory function on cellular transcription, and subsequently modulate cellular antiviral state and/or induce cell death. Regarding the pathogenesis in SBV-infected foetuses, nucleolar stress could be responsible for neurons degeneration and subsequent developmental abnormalities. At molecular level, our aim is to define the role of nucleolar protein B23 on viral replication, which is strongly relocalised to the nucleoplasm during SBV infection. Finally, hypothesis of NSs retrograde transport from cell cytoplasm to nucleus and the possible contributions of MVP and/or Tctex-1 needs to be further investigate.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, an opportunistic but serious human disease endemic to Southeast Asia and Northern Australia. The ‘Bp-group’ includes Bp and the closely-related organisms B. thailandensis (Bt) and B. oklahomensis (Bo), all of which are usually soil-dwelling saprophytes, and B. mallei (Bm) which is an equine-host-adapted pathogen. Bt is virulent in a number of invertebrate models but is generally non-pathogenic for mammals and is often used as a surrogate for the study of virulence mechanisms shared with Bp. Experiments to assess the potential of the Tobacco Hawkmoth Manduca sexta as a model host for Bp or Bt infection revealed surprising results. Bp, Bt and Bo were all lethal to M. sexta larvae. This is the first report of Bo virulence in an infection model. Additionally, the relative virulence of the three species was the reverse of that reported in humans and in larvae of the Greater Waxworm Galleria mellonella. Despite that, well-known hallmarks of Bp-group pathogenesis in mammalian hosts – intracellular survival and multiplication, actin remodelling and acute sepsis – were observed in M. sexta infection during a fluorescent confocal microscopy time-course study. M. sexta feeding experiments with Bt and Bo indicated that cultures of these bacteria are also pathogenic via the oral route, which is likely to be relevant for natural insect-bacteria interactions. Cell-free supernatant of Bo was as harmful to larvae as complete culture, supporting previous suggestions that Bp-group bacteria produce toxins or paralytic agents that are active against invertebrates. Finally, Rapid Virulence Annotation (RVA) was performed as a genome-wide screen for virulence determinants of Bp strain K96423, using three invertebrate bioassays with a recombinant expression library. In response to problems with the reproducibility of biologically active clones, a new statistical approach was devised which enabled quantitative identification of the most convincing RVA hits.

Les parasites Leishmania sont les agents responsables de la leishmaniose viscérale (LV), mucocutanée (LM) ou cutanée (LC) chez l'homme et de la leishmaniose canine (CanL). Ces parasites sont phagocytés par les macrophages humains et animaux où ils vont se transformer de promastigote en amastigote. Nous avons identifié et analysé les lipides impliqués dans le processus de transformation dans le complexe de Leishmania donovani et infantum. Quatre classes de lipides, les phospholipides, les acides gras libres, les triglycérides et les stérols ont été étudiés. De même, nous avons analysé la composition en acides gras des lipides totaux chez neuf espèces de Leishmania isolées en Tunisie, dont quatre souches de L. infantum, deux souches de L. major et deux souches de L. tropica et une souche de L. infantum de chien. Durant nos premiers travaux, la composition en acides gras des lipides totaux a été analysée et nos résultats montrent une augmentation des acides gras et du cholestérol et une diminution des triglycérides et de l'ergostérol durant la transition entre les promastigotes et les amastigotes. En ce qui concerne les classes de phospholipides, nous avons trouvé une proportion accrue de sphingomyéline et de phosphatidylsérine et une proportion réduite de phosphatidylinositol et de lysophosphatidyléthanolamine. Pour la composition en acides gras, une augmentation significative des acides gras n-7 a été observée chez les amastigotes, quant aux acides gras n-6 totaux, ils ont diminué chez les PL. Plusieurs changements ont également été observés au niveau des TG et des acides gras libres, en particulier, les acides gras n-7 et 20: 4n-6 ont été fortement augmentés, alors que les acides gras n-9 et les précurseurs n-6 ont diminué. Pour la deuxième étude, les résultats trouvés dans nos premiers travaux qui concernent la présence de proportions très élevés de 18 : 2n-6 contre de faible proportion de l’AA (20 :4n-6) ainsi que l’absence ou la très faible proportion de 18 :3n-3 ont été confirmés. De plus, L. major présentait une proportion plus élevée de 14 : 0 (acide myristique), 18: 3n-6 (acide gamma-linoléique) et une plus faible proportion de n-3, y compris 18: 3n-3 (acide alphalinoléique) et 22: 6n-3 (acide docosahexaénoïque) comparé à L. infantum et L. tropica. Après la supplémentation de l'acide oléique (AO), l'acide arachidonique (AA) et l’acide docosahexaénoïque (DHA) sur deux souches de L. infantum utilisées pour infecter les macrophages : un isotype MON-24 responsable de la leishmaniose cutanée et un MON-1 causant la leishmaniose viscérale ; nous avons constatés que les AA et DHA augmentaient la virulence du parasite, tandis que L’AO diminuait leur virulence / Leishmania parasites are the causative agents of visceral (LV), mucocutaneous (LM) or cutaneous (LC) leishmaniasis in humans, canine leishmaniasis in dogs (CanL), and leishmaniases in other mammals .The parasites are phagocytosed by human and animal macrophages where they will transform from promastigote to amastigote. We identified and analyzed the lipids involved in the transformation process in the Leishmania donovani complex. Four classes of lipids, phospholipids (PL), free fatty acids (FA), triglycerides (TG) and sterols were studied.Similarly, we analyzed the fatty acid composition of total lipids in nine Tunisian Leishmania isolates, including five strains of L. infantum (four human and one canine), two strains of L. major and two strains of L. tropica.In our early work, the fatty acid composition of total lipids was analyzed and our results show an increase in fatty acids and cholesterol and a decrease in triglycerides and ergosterol during the transformation of promastigotes into amastigotes. For phospholipid classes, we found an increase in sphingomyelin and phosphatidylserines and a decrease in phosphatidylinositols and lysophosphatidylethanolamines during processing. For the fatty acid composition, a significant increase of n-7 fatty acids was observed in amastigotes, as for n-6 total fatty acids, they decreased in PLs. Several changes have also been observed in TG and free fatty acids, in particular, n-7 and 20: 4n-6 fatty acids have been greatly increased, whereas n-9 fatty acids and n-6 ??precursors have been significantly increased. decreased.The study of the fatty acid composition of the 9 Tunisian strains of Leishmania confirmed, in the first place, our results found in our first works concerning the presencevery high proportions of 18: 2n-6 against low proportion of AA (20: 4n-6) as well as the absence or very low proportion of 18: 3n-3.In addition, the comparison of the fatty acid compositions of the three species studied: L. infantum, L. major and L. tropica showed that L. major had a higher proportion of 14: 0 (myristic acid), 18: 3n- 6 (gamma-linoleic acid) and a lower proportion of n-3, including18: 3n-3 (alpha-linoleic acid) and 22: 6n-3 (docosahexaenoic acid) compared to L. infantum and L. tropica. After supplementation of oleic acid (AO), arachidonic acid (AA) and docosahexaenoic acid (DHA) on two L. infantum strains used to infect macrophages: a MON-24 isotype responsible for cutaneous and a MON-1