Дисертації з теми "Type III secretion helper proteins"

Ralstonia solanacearum, l'agent causal du flétrissement bactérien, est considéré comme l'un des agents pathogènes bactériens les plus importants au monde. Le pouvoir pathogène de cette bactérie du sol est principalement basé sur son système de sécrétion de type III (SST3) et ses effecteurs de type III (ET3s), provoquant la maladie sur plus de 250 espèces végétales. R. solanacearum injecte ses ET3s à travers cette seringue moléculaire directement à l'intérieur de la plante hôte. Ces ET3s détournent les réponses de défense de la plante, soit dans le cytoplasme, soit dans le noyau, afin de supprimer l'immunité de la plante et de favoriser la multiplication bactérienne. La sécrétion des ET3s est finement contrôlée au niveau post-traductionnel par des protéines associées au contrôle de la sécrétion de type III, et par des protéines chaperonnes de type III.A ce jour, la fonction in planta de ces effecteurs et protéines chaperonnes, et la manière dont R. solanacearum module l’immunité de la plante en sa faveur restent mal comprises. Mon projet de thèse visait à mieux comprendre le rôle des déterminants de la pathogénicité de R. solanacearum en identifiant certaines des cibles d’A. thaliana, directes ou indirectes, modulées par la bactérie. Pour ce faire, j'ai utilisé des populations naturelles d'A. thaliana à deux échelles géographiques et adopté l'approche consistant à confronter des populations naturelles à des mutants de R. solanacearum dans lesquels des déterminants majeurs de pathogénie sont mutés. Cette approche est nouvelle puisque à ce jour les études d’association pangénomique (GWAS) menées sur les interactions plantes-agents pathogènes utilisent des souches sauvages de phytopathogènes. En outre, cette approche a permis de découvrir une diversité de réponses qui n'avaient pas été détectées auparavant. Dans la première partie de mon projet de thèse, j'ai identifié des QTLs impliqués dans la résistance quantitative à la maladie en réponse à des mutants simples de R. solanacearum, et j'ai validé fonctionnellement ces QTLs (Quantitative Trait Loci) comme facteurs de sensibilité. Dans la deuxième partie de ma thèse, nous avons étudié un gène codant pour une protéine de type NLR que nous avons appelé Bacterial Wilt Susceptibility 1 (BWS1). Nous avons montré que BWS1 agissait comme un facteur de sensibilité quantitatif, ayant un rôle de régulateur négatif dans une réponse immunitaire dépendante de SGT1.)
Ralstonia solanacearum, the causal agent of bacterial wilt, is considered one of the world’s most important bacterial pathogens. This soil-borne bacterium relies mainly on its type III secretion system (T3SS) and type III effectors (T3Es) in order to cause disease in more than 250 plant species. R. solanacearum injects its T3Es through this molecular syringe directly inside the host plant. These T3Es hijack plant defense responses in either the cytoplasm or the nucleus aiming to suppress plant immunity and promote bacterial multiplication. T3E secretion is finely controlled at the post-translational level by helper proteins, called T3SS control proteins, and type III chaperones.To date, the in planta function of these effectors and helper proteins and how R. solanacearum modulates plant genes to its favor remains poorly understood. My thesis project aimed to better understand the role of R. solanacearum pathogenicity determinants by identifying some of the direct or indirect plant targets of A. thaliana, modulated by the bacterium. For this purpose, I used natural populations of A. thaliana on two geographical scales and adopted the approach of challenging mapping populations to R. solanacearum mutants in which major pathogenic determinants are mutated. This approach is new since most of the GWAS (Genome-Wide Association Studies) in plant-pathogen interactions use wild-type strains of phytopathogens. Furthermore, it unveiled a previously undetected diversity of responses. In the first part of my Ph.D. project, I identified QTLs (Quantitative Trait Loci) involved in quantitative disease resistance to R. solanacearum single mutants and I validated these QTLs as susceptibility factors. In the second part of my thesis, we studied a gene encoding for a NLR protein that we called Bacterial Wilt Susceptibility 1 (BWS1). We showed that BWS1 was acting as quantitative susceptibility factor, mediating a negative regulation of an SGT1-dependent immune response

Type-III secretion systems (T3SSs) are responsible for the biosynthesis of flagella, and the interaction of many animal and plant pathogens with eukaryotic cells. T3SSs consist of multiple proteins which assemble to form an apparatus capable of exporting proteins through both membranes of Gram-negative bacteria in one step. Proteins conserved amongst T3SSS can be used for analysis of these systems using computational homology searching. By using tools including BLAST and HMMER in conjunction phylogenetic analysis this thesis examines the range of T3SSs, both in terms of the proteins they contain, and also the bacteria which contain them. In silico analysis of several of the conserved components of T3SSs shows similarities between them and other secretion systems, as well as components of ATPases. Use of conserved components allows for identification of T3SS loci in diverse bacteria, in order to assess in the different proteins used by different T3SSs, and to see where, in evolutionary space, these differences arose. Analysis of homology data also allows for comprehensive re-annotation of T3SS loci within Desulfovibrio, Lawsonia and Hahella, and subsequent comparison of these T3SSs with related Yersinial T3SSs, and also (in conjunction with in vitro assays) for identification of many novel effectors in E. coli.

Chlamydia trachomatis est une bactérie intracellulaire obligatoire. Ce pathogène de l’Homme est la première cause infectieuse de cécité ainsi que de maladies sexuellement transmissible d’origine bacterienne.Utilisant une souche de C. trachomatis L2 exprimant une protéine fluorescente, nous avons développé des méthodes de microscopie et de cytométrie en flux permettant de suivre les différentes étapes du développement de la bactérie. Ces méthodes faciliteront les futures études de l’infection par Chlamydia.Chlamydia interagit avec différents processus cellulaires, et plus particulièrement via la sécrétion d’effecteurs par le système de sécrétion de type 3 (ST3). Nous avons identifié une famille de protéines possédant un signal de ST3 qui partagent un domaine, le DUF582, présent uniquement chez les Chlamydia pathogènes.Nous avons montré que les 5 protéines DUF582 de C. trachomatis sont exprimées à partir du milieu du cycle infectieux. Nous avons démontré que la protéine Hrs interagit avec le DUF582 et que la protéine DUF582 CT619 interagit avec Tsg101. Hrs et Tsg101 sont d’importants composants de la machinerie ESCRT impliquée dans de nombreux processus de fission membranaire.Utilisant l’interférence ARN, nous avons montré que Hrs et Tsg101 ne sont requis ni pour l’entrée, ni pour le développement de la bactérie. Ceci suggère que les protéines DUF582 bloquent des processus dépendant de Hrs/Tsg101. A l’inverse, la bactérie pourrait utiliser la machinerie ESCRT mais l’existence de mécanismes redondants expliquerait l’absence de phénotype dans les expériences d’interférence. Nous discutons trois hypothèses concernant le rôle des protéines DUF582 dans l’infection
Chlamydia trachomatis is an obligate intracellular human pathogen. It is the first infectious cause of blindness and the most common cause of sexually transmitted diseases of bacterial origin. Using a strain of C. trachomatis serovar L2 expressing a fluorescent protein we developed microscopy and flow cytometry based methods to quantify several steps of its developmental cycle. These methods will facilitate future studies aimed at testing anti-bacterial compounds or various culture conditions. Chlamydiae interfere with many cellular processes, in particular via the secretion of bacterial proteins through a type 3 secretion (T3S) system. We identified a family of proteins that possess T3S signals. They share a domain designated as DUF582, which is only found in pathogenic chlamydiae. We showed that the five DUF582 proteins of C. trachomatis are expressed from the mid phase of infection. We demonstrated that the protein Hrs is a common interactor for the DUF582. In addition the N-terminal part of the DUF582 protein CT619 interacts with Tsg101. Hrs and Tsg101 are both important components of the ESCRT machinery, which is an ancient machinery required for several processes involving membrane fission.Using RNA interference we showed that Hrs and Tsg101 are dispensable for bacterial entry and growth. This last result suggest that DUF582 proteins actually prevent Hrs and/or Tsg101 driven processes. Alternatively, the bacteria might highjack the ESCRT machinery but redundant mechanisms would explain the absence of phenotype on bacterial development observed in the silencing experiments. We discuss three hypotheses as to the possible role of the DUF582 proteins in infection

Xanthomonas campestris pv. campestris (Xcc) est responsable de la nervation noire sur les Brassicaceae, notamment les choux, les radis, la moutarde et l'espèce modèle Arabidopsis thaliana. Au cours de l'infection, Xcc transloque des protéines effectrices de type 3 (T3E) dans les cellules végétales via le système de secrétion de type 3 (T3SS), pour moduler la physiologie de l'hôte et favoriser la maladie. Le répertoire des T3E présents dans une souche donnée influence largement sa niche, sa gamme d'hôtes et son mode de vie. Dans la souche Xcc 8004, vingt-huit gènes ont été prédits pour coder des protéines sécrétées par le T3SS (T3SP). Dans un premier chapitre à l'échelle de l'effectome, nous montrons que la plupart des fonctions des T3SP au sein des cellules végétales restent inconnues. Dans ce projet, différentes stratégies ont été abordées pour caractériser les fonctions biologiques des T3SPs de la souche Xcc 8004 dans les cellules végétales. Bien que la délétion des gènes individuels codant des T3SP n'ait pas eu d'effet significatif sur la virulence de Xcc chez Arabidopsis, l'expression hétérologue de T3SP individuelles chez Arabidopsis a montré des effets marqués sur la physiologie de la plante pour de nombreuses T3SP. De manière surprenante, plusieures T3SP sont capables de déclencher des réponses immunitaires et certaines ont présenté des effets ambivalentes en inhibant simultanément la phosphorylation déclenchée par flg22 de MPK3/6. Dans une deuxième partie, nous avons réalisé une analyse comparative des fonctions in planta des T3SPs XopAG et RipO1 qui sont codées par des gènes orthologues dans Xcc souche 8004 et Ralstonia solanacearum souche GMI1000, respectivement. Dans nos expériences, XopAG a montré une contribution significative à la pathogénicité de Xcc qui ne semble pas liée à la suppression de certaines réponses immunitaires basales. XopAG et RipO1 présentent des similitudes fonctionnelles. En effet, les deux T3SP affectent l'expression de gènes connus de réponse à l'auxine, à l'acide jasmonique et à l'éthylène, ce qui est cohérent avec l'inhibition de la croissance des plantes induite par ces deux effecteurs chez Arabidopsis. Une recherche in silico de cibles végétales potentielles de XopAG suivie d'essais du pouvoir pathoène sur des mutants d'Arabidopsisa a permis d'identifier BRG3 (BOI-RELATED GENE 3) comme cible putative de XopAG. Dans une approche parallèle, nous avons effectué un criblage de suppresseurs pour identifier les mutations qui atténuent le phénotype d'arrêt de croissance induit par XopAG chez Arabidopsis, résultant en l'identification de huit lignées suppresseurs. Ces lignées fournissent une occasion précieuse d'identifier les voies affectées par XopAG chez Arabidopsis. De manière générale, ce projet contribue à la compréhension des activités biologiques exercées par les T3SP de la souche Xcc 8004 dans les cellules végétales
Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on Brassicaceae species including cabbages, radish, mustard and the model species Arabidopsis thaliana. During pathogenesis, Xcc secrete Type 3 Effector (T3E) proteins via the Type 3 Secretion System (T3SS) into plant cells to modulate host physiology and promote pathogenicity. The repertoire of T3Es present in a given strain largely influences its niche, host range and lifestyle. In the Xcc strain 8004, twenty-eight genes have been predicted to encode proteins secreted by the T3SS. The functions of most Type 3 Secreted Proteins (T3SPs) within plant cells remain elusive. In this project, different strategies were approached to characterize the biological functions of the T3SPs of Xcc strain 8004 in plant cells. In the first chapter, we showed that the loss of individual T3SPs did not cause a significant effect on Xcc virulence on Arabidopsis. Yet, the heterologous expression of individual T3SPs in Arabidopsis plants revealed many T3SPs with marked effects on plant growth and transcriptome. Several T3SPs also triggered plant immune responses and some exhibited ambivalent activities by simultaneously inhibiting flg22-triggered phosphorylation of MPK3/6. In the second chapter, we conducted a comparative analysis of the in planta functions of the T3E XopAG and RipO1 which are encoded by orthologous genes in Xcc strain 8004 and Ralstonia solanacearum strain GMI1000 respectively. In our experiments, XopAG showed a significant contribution to Xcc pathogenicity that was not related to the suppression of some basal immune responses. XopAG and RipO1 exhibited functional similarities. Indeed, both T3E affected the expression of genes responsive to auxin, jasmonic acid and ethylene suggesting that both effectors inhibit plant growth. Finally, we made some efforts to identify the plant target of XopAG. An in silico search followed by pathogenicity assays posits BRG3 (BOI-RELATED GENE 3) as a candidate target of XopAG. In a parallel approach, we performed a suppressor screen to identify suppressor mutations that alleviate the growth defect induced by XopAG in Arabidopsis plants, resulting in eight suppressor lines. These provide a valuable opportunity to identify the pathways targetted by XopAG in Arabidopsis. Altogether, this project contributes to the better comprehension of the biological activities exerted by the Xcc strain 8004 T3SPs in planta

The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine (NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

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O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris
Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system’s secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system’s proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system’s pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris

Resumo: O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris
Abstract: Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system's secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system's proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system's pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris
Orientador: Jesus Aparecido Ferro
Coorientador: Julio Cezar Franco de Oliveira
Banca: Maria Teresa Marques Novo
Banca: Leandro Márcio Moreira
Mestre

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

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

Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.

Chlamydia trachomatis remains a highly relevant clinical pathogen as it is the causative agent of the most commonly reported sexually transmitted disease in the western hemisphere, and the most common cause of infectious blindness in the developing world. As an obligate intracellular pathogen, Chlamydia employs a vast assay of virulence proteins to hijack and remodel the host cellular machinery to facilitate its growth and dissemination. Besides delivering effector proteins into the host cytoplasm via a conserved type III secretion machinery, Chlamydia encodes components of multiple secretion systems, such as type II and IV. Chapter 3 of this document describes the secretion, processing and localization of two putative autotransporters (Pls1 and Pls2) and their involvement in inclusion expansion.

In recent years, many new chlamydial effector proteins have been described. CPAF (Chlamydial Protease-like Activity Factor) is a secreted serine protease that is emerging as a central virulence protein: it is proposed to play a central role in Chlamydia pathogenesis by cleaving proteins involved in antigen-presentation, apoptosis and cytoskeletal re-arrangements. However, the functional significance of CPAF remains elusive due to the lack of specific inhibitors and Chlamydia mutants. The body of work presented herein demonstrates that in addition to targeting host proteins, CPAF cleaves a subset of early chlamydial effector proteins, including Inc-proteins that reside on the nascent pathogenic vacuole ("inclusion"). The design and development of a CPAF-specific inhibitory peptide demonstrates that these chlamydial effector proteins are true targets of CPAF. This peptide reversed the cleavage of bacterial targets by CPAF both in an in vitro cleavage assay and during infection, indicating that these effectors are bona fide targets. Inhibition of CPAF activity also revealed that this protease regulates multiple facets of chlamydial pathogenesis. CPAF inhibition in infected epithelial cells led to the complete dismantling of the inclusion, secretion of pro-inflammatory cytokines and engagement of an inflammasome-dependent programmed cell death pathway. While fibroblasts defective in various inflammasome components were resistant to Chlamydia-induced cell death, inclusion integrity and bacterial replication was still compromised upon CPAF inhibition, indicating that loss of inclusion integrity was not a consequence of caspase-1 activation. Overall, these findings revealed that CPAF, in addition to regulating host function, directly modulates the activity of secreted effectors and early Inc-proteins. Furthermore, we establish that CPAF is an essential virulence factor that is required to maintain the integrity of the inclusion and prevent the engagement of innate immune programmed cell death pathways in infected epithelial cells. CPAF activity thus remains a compelling mechanism by which intracellular pathogens employ proteolytic events to modify the host environment.

Dissertation