Pepper is a popular and important vegetable crop grown and consumed worldwide. However, pepper production is threatened by the gram-negative bacterium Xanthomonas euvesicatoria (Xe) which causes bacterial spot (BS) disease, one of the most common and destructive diseases on pepper. Due to limited genetic resistance resources in host species, a promising strategy for controlling BS disease is to transfer nonhost disease resistance (R) genes from other plant species into pepper plants to confer broad-spectrum and durable resistance. A maize R gene Rxo1 has been functionally transferred to rice plants and confers nonhost resistance to rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc) carrying a type III effector (T3E) AvrRxo1. Most Xe strains carry a T3E Xe4428, a homolog of AvrRxo1. Therefore, Rxo1 could be potentially employed to develop Xe-resistant pepper. In addition, a better understanding of the virulence function of Xe4428 may provide insights into the pathogenesis of Xe and new strategies for crop improvement. In this dissertation, we transformed Rxo1 into the far-related dicot species Nicotiana benthamiana and pepper, and characterized the Rxo1-mediated disease resistance against Xe strains carrying AvrRxo1 or Xe4428. In addition, we explored the virulence function and mechanism of Xe4428. In the Rxo1-transgenic N. benthamiana, we demonstrated that Rxo1 could condition resistance to Xe harboring AvrRxo1 but not Xe4428. We revealed that AvrRxo1 could directly interact with the nucleotide-binding domain of Rxo1 in vivo and in vitro. We further demonstrated that the nucleus localization of AvrRxo1 was required for its avirulence and virulence functions. In addition, the cytosol localization of Rxo1 was also necessary to confer disease resistance. The downstream signaling component NbNDR1 was demonstrated to be involved in Rxo1/AvrRxo1-mediated disease resistance. By RNAseq-based gene expression profiling, we identified six candidate genes of interest up-regulated by the Rxo1-AvrRxo1 recognition. Through virus-induced gene silencing screening, a gene encoding phenylalanine ammonia-lyase 4 was demonstrated to be critical for Rxo1/AvrRxo1-mediated disease resistance in N. benthamiana. Rxo1-transgenic pepper plants were resistant to the Xe strain with the complementary Xoc effector AvrRxo1 but not the wild-type Xe strain that carries Xe4428. A Xe4428 mutant with only one nucleotide substitution could trigger the Rxo1-mediated disease resistance in pepper. Both wild-type and mutant Xe4428 had significant virulence functions that could promote the Xe bacterial proliferation on wild-type pepper plants. In addition, the mutant Xe4428 had a higher expression level than wild-type Xe4428 in Xe bacterial cells, which might explain why the mutant Xe4428 but not wild-type Xe4428, could trigger the Rxo1-mediated disease resistance in pepper. We identified 14 pepper cystatin genes (CaCys), among which two genes (CaCys1 and CaCys13) could be induced, and two genes (CaCys3 and CaCys5) were suppressed by Xe4428. Ectopically expressing one of the induced genes CaCys1 in N. benthamiana increased the stomatal opening and promoted the Xe growth in N. benthamiana plants. Thus, we illuminate one possible mechanism of Xe4428's virulence function is to regulate the stomata apertures in N. benthamiana. Bacterial fruit blotch (BFB) caused by the gram-negative bacterial pathogen Acidovorax citrulli (A. citrulli) is one of the most destructive diseases in cucurbit crops, including melon and watermelon. A better understanding of the virulence and avirulence functions of T3Es in A. citrulli helps breeders engineer crop resistance to BFB. To this end, a clean genetic background of A. citrulli with multiple effector genes deleted is desired. Here, we optimized a marker-exchange-based method for sequential effector deletion and generated an AAC00-1 mutant with five effector genes (Aave2166, Aave3626, Aave1548, Aave2938, Aave2708) deleted (AAC00-15). AAC00-15 was less virulent in watermelon but more virulent in N. benthamiana. Through complementation, we characterized the function of individual effectors and identified a promising R gene, Roq1, that could be used to control BFB disease. / Doctor of Philosophy / As an essential ingredient in almost all cuisines, pepper is grown and consumed worldwide, providing human beings with favorable flavor and nutrients. However, pepper production is threatened by the destructive bacterial spot (BS) disease caused by the bacterial pathogen Xanthomonas euvesicatoria (Xe). Due to limited genetic resistance resources in host species, nonhost resistance (R) genes from other plant species are desired to confer broad-spectrum and durable resistance to the pepper pathogen Xe. Previously, a maize (corn) R gene called Rxo1 was transferred to rice plants. This gene helped these rice plants resist a rice bacterial pathogen that causes leaf streak disease on rice. This rice pathogen has an effector (a virulent protein produced by bacteria to infect plants) that is required for the disease resistance. The pepper pathogen carries a similar effector, so transferring the maize R gene Rxo1 to pepper plants might similarly benefit peppers and help fight against the bacterial spot disease. In this dissertation, we successfully transferred the maize R gene Rxo1 into Nicotiana benthamiana and pepper plants. Our results indicate that this gene can help control disease caused by the pepper pathogen harboring the effector of the rice pathogen but not its native effector. We also illuminate how the disease resistance conferred by this maize gene happens in Nicotiana benthamiana plants. In addition, we explain how the corresponding effector helps infect plants. This research provides insights into the application of R gene transfer between far-related plant species and new tools to improve crop disease resistance.

Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells. / Doctor of Philosophy / Peppers and tomatoes are two of the most important vegetables grown worldwide, providing humans with high quality of flavor and aroma, vitamins, and antioxidants. The pepper and tomato production is frequently threatened by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on peppers and tomatoes. The BS disease can be easily identified due to the appearance of the dark, irregular, water-soaked areas on the leaf, which can cause approximately 10% loss of the total yield of peppers and tomatoes. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most critical breeding goals. A better understanding of the virulence mechanism of Xe could help breeders to design new strategies for resistance breeding. In my seminar, I will discuss the virulence and avirulence functions of Xe type three secretion (T3S) effectors: Xe XopN, Xe XopQ, and Xe XopX. In my study, I identified Xe XopN is a key factor that regulates the development of the water-soaking symptom on pepper plants infected with Xe. In addition, we revealed Xe XopN interacts with a transcription factor NbVOZ to regulate the expression of NbNPR1 and PR1 genes expression, which may also contribute to the development of water-soaking phenotype. In addition, I identified that Xe XopN could interact with a transcription factor, NbVOZ, and represses the expression of NbNPR1, a key component of the basal defense, and the pathogenesis-related gene PR1. Therefore, Xe XopN has a role in regulating a water-affluent environment to promote bacterial proliferation in the infected plant tissue. Xe XopQ is a Xe T3S effector that functions as a determinant of host specificity. In my study, I identified another T3S effector Xe XopX that could interact with Xe XopQ to trigger the defense response in Nicotiana benthamiana. I also confirmed Xe XopQ physically interacts with Xe XopX inside of plant cells by using bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, Xe XopX could also interact with multiple Xe T3Es including AvrBS2 in a co-IP assay. The virulence and avirulent functions of Xe XopQ and AvrBS2 are compromised in the absence of Xe XopX.

The Type III secretion system (T3SS) of Pseudomonas aeruginosa uses a needle-like protein apparatus to detect eukaryotic host cells and translocate effectors directly into the host cell. The effectors are also known as cytotoxins, which cause disruption of a series of signaling events in the host cell, facilitating the infection by P. aeruginosa. As the T3SS is antigenic and the expression of T3SS is energy-consuming, it is highly regulated where several regulatory proteins interact with each other and control the expression of T3SS genes. Among these proteins, ExsA, the master regulator of T3SS in P. aeruginosa, is of great importance as it is a transcriptional activator that activates the expression of all T3SS genes. Also, as ExsA belongs to the AraC protein family which only exists in bacteria and fungi, it makes an excellent potential target for drugs against P. aeruginosa related infections. With a combination of molecular biology tools and structural biology methods, we solved the N-terminal domain structure of the ExsA protein in P. aeruginosa. The model of the ExsA N-terminal domain has enriched our knowledge about ExsA dimerization and can serve as the base for mapping the interaction interfaces on ExsA and ExsD. Further, we have found two homologues of ExsA by structural alignment, which share a lot of similarities and have conserved amino acid residues that are important for ligand binding. The fact that both of these two proteins are regulated by small ligands rather than proteins also raises the possibility that ExsA may have a second regulatory mechanism under which ExsA is regulated by a small ligand, which so far has not been observed or reported by researchers. In order to map the binding site of ExsA on its anti-activator ExsD, we removed the coiled-coil region (amino acid residue 138-202, the potential binding site) of ExsD, based on the  structure of ExsD. We surprisingly found that the ExsD variant without the coiled-coil region readily inhibits ExsA-dependent in vitro transcription. This result rules out other possibilities and makes us focus on the N-terminus and adjacent regions of ExsD for the interface with ExsA. Moreover, in order to gain a comprehensive understanding of the dynamics of the regulation of T3SS in P. aeruginosa, we have begun to build a mathematical model of the T3SS regulatory pathways. We are measuring the cellular concentrations of T3SS regulatory proteins with quantitative molecular biology methods such as quantitative western blot, quantitative PCR and quantitative mass spectrometry. We have determined the cellular level of ExsA and ExsD proteins under different physiological conditions, and found that some factors such as temperature have a significant impact on the levels of ExsA and ExsD. This study has thus unveiled some unknown features of the T3SS of P. aeruginosa and its related infections. / Master of Science

Yes / Interferon lambda (IFNλ) is important for epidermal defence against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signalling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of MxA, a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate STAT1 in fibroblasts, but instead activated MAPKs. Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of TGFβ1-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signalling and suggests that IFNλ, whilst important for epidermal anti-viral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

Xanthomonas oryzae pv. oryzae (Xoo) est une bactérie phytopathogène responsable de la bactériose vasculaire du riz, maladie pouvant engendrer de fortes pertes de rendement à travers le monde. La course à l'armement entre la bactérie et sa plante hôte correspond d'une part à la mise en place de la virulence par le microorganisme et d'autre part en la résistance du végétal face à l'agression. Comprendre les mécanismes par lesquels Xoo accompli son cycle infectieux est d'une importance cruciale pour le développement futur de nouvelle méthode de luttes. Plusieurs approches complémentaires ont été mises en uvre afin de caractériser des éléments associés au pouvoir pathogène de Xoo.Dans un premier temps nous avons effectué une analyse protéomique comparative. Cette approche a permis l'identification chez une souche Africaine de Xoo d'un jeu de protéines induites par HrpX et susceptibles de jouer un rôle dans la virulence. Dans un second temps, l'implication de deux peptides dans la virulence Xoo a été étudiée. Le premier de ces peptides, supposé être le facteur d'avirulenceAvrXa21, a fait l'objet d'une caractérisation fonctionnelle et phylogénique. Le second peptide est synthétisé par un cluster NRPS, similaire à l'un de ceux présent chez Xanthomonas albilineans. Afin d'élucider l'importance de la molécule synthétisée par cette voie pour Xoo, une étude préliminaire impliquant la mutation d'un élément régulateur des NRPS a été effectuée. En dernier lieu, des informations nouvelles ont été apportées sur la topologie de la protéine membranaire HrcR qui est une composante essentielle du système de sécrétion de type III chez la plupart des bactéries appartenant au genre Xanthomonas. / Xanthomonas oryzae pv. oryzae (Xoo) is the agent of bacterial leaf blight BLB in rice, a disease which causes considerable yield losses throughout the world. In the arms race underlying the interactions between the microorganism and the host, the presence of virulence factors in the former parallels that of resistance factors in the latter. Understanding the mechanisms of Xoo's infectious cycle is of paramount importance for the elaboration of new fighting strategies to combat BLB. To achieve this, several complementary approaches to characterize components of Xoo's pathogenicity have been employed.First, we performed comparative proteomics that allowed us to identify novel HrpX-induced candidate pathogenicity factors of an African Xoo strain. Second, the involvement of two peptides in Xoo's pathogenicity has been investigated. One was speculated to be the avirulence factor AvrXa21 and has been characterized both functionally and phylogenetically. The other one was found to be synthesized by a Non-Ribosomal Peptide Synthetase (NRPS), reminescent to NRPS genes found in Xanthomonas albilineans. In order to determine the role of NRPS-mediated synthesis in Xoo virulence, we studied a strain carrying a mutated regulatory gene of the NRPS pathway. Finally, we provide new information on the topology of the HrcR membrane protein which is a conserved component of the type III secretion system of most Xanthomonas.

Cette thèse est une étude théorique d’un système d’équations de Cahn-Hilliard/Allen-Cahn couplées qui représente un mélange binaire en séparation de phase. Le but principal de l’étude est le comportement asymptotique des solutions en termes d’attracteurs exponentiels/globaux. Pour cette raison, l’existence et l’unicité de la solution sont étudiées tout d’abord. Une des principales applications de ce modèle d’équations est la cristallographie.Dans la première partie de la thèse, on examine le modèle proposé avec des conditions de type Dirichlet sur le bord et une non linéarité régulière de type polynomial : on réussit à trouver un attracteur exponentiel et par conséquence un attracteur global de dimension finie. Une non linéarité singulière de type logarithmique est ensuite prise dans la deuxième partie, cette fonction étant approchée par une suite de fonctions régulières et l’existence d’un attracteur global est démontrée sous des conditions au bord de type Dirichlet.Enfin, dans la dernière partie, le système est couplé avec une équation pour la température: suivant la loi de Fourrier premièrement, puis la loi de type III de la thermo-élasticité. Dans les deux cas, la dynamique de l’équation est étudiée et un attracteur exponentiel est trouvé malgré la difficulté créée par l’équation hyperbolique dans le deuxième cas. / This thesis is a theoretical study of a coupled system of equations of Cahn-Hilliard and Allen-Cahn that represents phase separation of binary alloys. The main goal of this study is to investigate the asymptotic behavior of the solution in terms of exponential/global attractors. For this reason, the existence and unicity of the solution are first studied. One of the most important applications of this proposed model of equations is crystallography. In the first part of the thesis, the system is studied with boundary conditions of Dirichlet type and a regular nonlinearity (a polynomial). There, we prove the existence of an exponential attractor that leads to the existence of a global attractor of finite dimension. Then, a singular nonlinearity (a logarithmic potential) is considered in the second part. This function is approximated by a sequence of regular ones and a global attractor is found.At the end, the system of equations is coupled with temperature: with the Fourrier law in the first case, then with the type III law (in the context of thermoelasticity) in the second case. The dynamics of the equations are studied and the existence of an exponential attractor is obtained.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012

La protéine BamB est une lipoprotéine de membrane externe appartenant au complexe BAM (β-Barrel Assembly Machinery) et impliquée dans l’assemblage des protéines de membrane externe (PME), la sensibilité aux antibiotiques, le contrôle de l’expression des trois systèmes de sécrétion de type III (T3SS) et la virulence de Salmonella. Chez E. coli, au sein du complexe BAM, elle interagit directement avec la protéine BamA. De plus, chez cette bactérie, BamB présente une activité sérine-thréonine kinase. Afin de mieux caractériser le rôle de BamB, nos objectifs ont été d’étudier (1) l’impact de l’altération de l’interaction de BamB avec le complexe BAM ou de sa séquestration dans le cytoplasme sur l’ensemble des rôles décrits de BamB et (2) l’activité kinase putative de BamB chez Salmonella. Nos résultats montrent que certains rôles de BamB sont dissociables entre eux et que l’interaction BamA/BamB n’est pas requise pour le rôle de BamB dans le contrôle de l’expression des T3SS, la virulence de Salmonella et l’assemblage des PME à la membrane externe. Aucune activité kinase ni aucune activité cytoplasmique de la protéine n’a pu être formellement démontrée. / BamB is an outer-membrane lipoprotein belonging to the BAM complex (β-Barrel Assembly Machinery). In Salmonella, it is involved in the assembly of outer membrane proteins (OMP), in antibiotic susceptibility, in the transcriptional control of the three Type-Three-Secretion-Systems (T3SS) related genes and also in virulence. In E. coli, BamB interacts directly with the BamA protein. Moreover, BamB has been shown to have a serine-threonin kinase activity in this bacterium. In order to better characterize the roles of the BamB protein, our purposes were to study (1) the impact of the alteration of the interaction of BamB with the BAM complex or of its cytoplasmic sequestration and (2) its putative kinase activity in Salmonella. Our results show that some of the BamB roles are dissociable and that the BamA/BamB interaction is not required for T3SS expression, Salmonella virulence or OMP assembly in the outer membrane. Currently, neither a kinase activity nor a cytoplasmic activity has been clearly demonstrated for this protein.

Les bactéries du genre Shigella sont responsables de la shigellose, une maladie diarrhéique invasive du colon. L’entrée et la dissémination de Shigella à travers l’épithélium colique sont médiées par un système de sécrétion de type III (SST3) codé par un plasmide de virulence. Au sein de ce plasmide se trouve une région de 30-kb comportant les gènes impliqués dans l’entrée de la bactérie dans les cellules hôtes. Ces gènes sont regroupés en deux loci :le locus ipa-ipg qui code pour les protéines sécrétées et leurs chaperons ainsi que le locus mxi-spa codant pour les composants de l’appareil de sécrétion de type III (AST3), constitué d’un bulbe cytoplasmique, d’un corps basal transmembranaire et d’une aiguille se projetant au niveau extracellulaire. Ce système permet la sécrétion ordonnée et hiérarchique de différentes classes de protéines et la translocation de certaines d’entre elles (appelées effecteurs) dans le cytoplasme de la cellule hôte où elles interfèrent avec les voies de signalisation cellulaires. Avant le contact avec la cellule hôte, l’AST3 est inactif et verrouillé par les protéines IpaB et IpaD formant le complexe d’extrémité.<p>Chez Shigella, le gatekeeper MxiC séquestre les effecteurs au niveau du cytoplasme bactérien avant la transmission par l’aiguille du signal d’activation de la sécrétion mais les composants intermédiaires liant l’aiguille à MxiC restaient inconnus. Au cours de ce travail, nous avons montré que MxiC forme un complexe avec la sous-unité de la tige interne, MxiI, afin de bloquer l’entrée du canal de sécrétion et que cette interaction est conservée chez Yersinia et Salmonella. Nous démontrons que, suite au contact cellulaire, la dissociation de ce complexe facilite le switch de sécrétion des translocateurs aux effecteurs. Nos résultats révèlent également que MxiC est capable de s’associer au chaperon IpgC afin de réguler la sécrétion des translocateurs. De plus, nous avons identifié les domaines de MxiC engagés dans la régulation du SST3 et rapporté un nouveau rôle de MxiC dans l’échappement aux macrophage impliquant une possible inhibition de la voie apoptotique classique afin de promouvoir une pyroptose. Chez Shigella, IpaD gouverne la composition du complexe d’extrémité et est impliqué dans la régulation de la sécrétion. Nous avons développé une étude phénotypique de ses régions coiled-coil et centrale et montré que la composition du complexe d’extrémité permet de définir à la fois l’état d’inductibilité de l’AST3 et la sécrétion des effecteurs tardifs. Par ailleurs, notre étude fonctionnelle des domaines de MxiC et IpaD suggère que les capacités de Shigella à échapper au macrophage et à insérer un pore de translocation ne sont pas strictement couplées. <p>La dernière partie de ce travail s’est focalisée sur la caractérisation de la protéine Spa13 de Shigella. Nous avons découvert que le défaut de sécrétion du mutant spa13 est dû à l’instabilité de la sous-unité MxiH de l’aiguille et que Spa13 n’est pas sécrété par le SST3. Nos résultats indiquent également un rôle de Spa13 dans l’escorte de chaperons et l’activation de l’appareil d’exportation afin de promouvoir la sécrétion des substrats./Shigella is the causative agent of shigellosis, also known as bacillary dysentery, an invasive disease of the human colonic epithelium. During infection, Shigella uses a type III secretion system (T3SS) to penetrate enterocytes and to disseminate into the colonic epithelium, leading to destruction of the mucosal lining and shigellosis symptoms. Most of the virulence factors of Shigella are encoded by a large plasmid harboring a 30-kb region that is sufficient to promote bacterial entry into host cells. This entry region is organized in two loci, one corresponding to the the ipa-ipg genes encoding the secreted proteins and their cognate chaperones while the other encodes Mxi-Spa proteins that form the type III secretion apparatus (T3SA), consisting of a cytoplasmic bulb, a basal body spanning the bacterial envelope and a hollow needle. The T3SS allows the ordered and hierarchical secretion of effectors by inserting a translocation pore in the host cell membrane through which effector proteins are injected into the cytosol. Before host cell contact, the T3SA is inactive and plugged by the tip complex proteins IpaB and IpaD. <p>In Shigella, the gatekeeper MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle but the molecules involved in linking the needle and MxiC are unknown. We demonstrated that MxiC and the predicted inner-rod component MxiI form a complex plugging the T3SA entry gate and showed that this interaction is conserved in Yersinia and Salmonella. Dissociation of this complex seems to facilitate the switch in secretion from translocators to effectors upon host cell contact. Our results also revealed that MxiC binds to the chaperone IpgC to regulate translocators secretion. Moreover, we identified the domains of MxiC involved in the T3S regulation and reported a new role in macrophage escape by potential inhibition of the classical apoptosis to promote pro-inflammatory pyroptosis. <p>In Shigella, IpaD rules the composition of the tip complex and is involved in secretion control and translocon insertion. We therefore undertook a phenotypic analysis of its coiled-coil and central regions and showed that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Besides, our functional study on MxiC and IpaD domains suggests that Shigella abilities to escape macrophage vacuole and to insert the translocation pore are uncoupled.<p>The last part of this work is related to the characterization of the Spa13 protein of Shigella. We found that the secretion defect of the spa13 mutant is due to the instability of the needle component MxiH and that Spa13 is not a secreted substrat. Our results also support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch to promote substrates secretion. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.