P. aeruginosa est un pathogène opportuniste capable d'infecter un large spectre d'hôtes. Elle possède un vaste arsenal de facteurs de virulence. Le système de sécrétion de type III (SSTT) est un facteur de virulence majeur dont la régulation est complexe pour permettre une adaptation la plus précise possible de la bactérie au cours de l'infection. Nous nous sommes intéressés à déterminer le rôle potentiel de nouveaux acteurs de l'adaptation de P.aeruginosa au cours de l'infection. La porine OprF qui représente la protéine la plus abondante de la membrane externe de P. aeruginosa lui permettrait d'évaluer l'état d'activation du système immunitaire de son hôte afin d'adapter sa virulence. Chez P. aeruginosa, le tryptophane est le précurseur des kynurenines qui sont également produites par l'hôte à partir du tryptophane et qui, dans ce dernier contexte, sont des immunomodulateurs. Peu ou pas d'études ont été réalisées pour mettre en œuvre un éventuel rôle d'immunomodulation ou dans la virulence des kynurénines bactériennes. Dans un premier temps, nous nous sommes intéressés à un signal anciennement découvert au laboratoire et qui réprime l'expression du SSTT à haute densité bactérienne. Nous avons montré que ce signal exerce une régulation post-transcriptionnelle en plus d'une inhibition de la transcription des gènes du SSTT. Le métabolisme du tryptophane et de l'anthranilate semble être au cœur de ce processus de régulation. En inactivant des voies du catabolisme du tryptophane, nous avons montré que la production de ce signal dépend partiellement de la voie des kynurénines mais ne dépend pas ni des voies classiques du quorum sensing ni de l'opéron phnAB, impliqué dans la synthèse de l'anthranilate. Cependant, la voie des phénazines pourrait être impliquée dans la production de ce signal. Par CLHP couplée à la spectrométrie de masse, nous avons pu séparer des espèces moléculaires réprimant le SSTT et qui sont contenues dans ce signal, mais l'identification précise nécessite plus d'investigations. Dans un second temps, nous nous sommes intéressés aux kynurénines produites par la bactérie. Nous avons confirmé que P. aeruginosa produit des kynurénines et le gène kynA est le gène clé de la voie de synthèse de ces métabolites. En utilisant des fusions transcriptionnnelles, nous avons montré que le tryptophane et la kynurénine régulent positivement la production des kynurénines en agissant sur l'expression des gènes clés. D'autres parts, nous avons remarqué que la bactérie module l'activité de la voie métabolique des kynurénines issue du tryptophane en fonction de son état de croissance. Nous avons montré qu'au cours du dialogue interrègne bactérie/hôte, la voie des kynurénines de P. aeruginosa est stimulée par certains composants du système immunitaire. Grâce à un modèle d'infection pulmonaire aiguë, nous avons prouvé que les kynurénines produites par la bactérie sont importantes pour sa virulence. Selon notre hypothèse les kynurénines pourraient avoir une action sur la réponse immune, mais cela reste à déterminer. Dans un troisième temps, nous nous somme focalisés sur la porine OprF. Nous avons montré que la mutation ∆oprF est à l'origine d'une altération de la production mais vraisemblablement pas de la sécrétion des exotoxines du SSTT. Un ligand connu d'OprF, l'interféron gamma, module la voie des kynurénines. OprF pourrait donc avoir un rôle central dans les différents aspects de la régulation de la virulence. Nous avons donc produit des anticorps monoclonaux anti-OprF. Ces derniers se sont révélés capables de reconnaître spécifiquement la protéine OprF. Afin de vérifier l'efficacité de ces anticorps, des expériences de neutralisation de la bactérie in vitro puis in vivo seront réalisées. Mots clés : Pseudomonas aeruginosa, Système de Sécrétion de Type III, régulation, catabolisme du tryptophane, kynurénines, OprF. / P. aeruginosa is an opportunistic pathogen capable of infecting a wide host range. It possesses a large arsenal of virulence factors. The type III secretion system (TTSS) is a major virulence factor whose regulation is complex to allow the most accurate adaptation of the bacteria during infection. We were interested to determine the potential role of new actors in the adaptation of P. aeruginosa during infection. OprF represents the most abundant protein of the outer membrane of P. aeruginosa. This protein allows bacteria to assess the activation status of the host's immune system to adapt its virulence. In P. aeruginosa, tryptophan is the precursor of kynurenines that are also produced by the host from tryptophan and in the latter context, are immunomodulators. Little or no studies have been done to determine a possible role of bacterial kynurenines in immune modulation or virulence. Initially, we were interested in a signal previously discovered in the laboratory and which suppresses the expression of TTSS at high bacterial density. We have shown that this signal exerts a post-transcriptional regulation in addition to inhibition of TTSS genes transcription. The metabolism of tryptophan and anthranilate appears to be at the heart of this regulatory process. By inactivating pathways of tryptophan catabolism, we showed that production of this signal depends partly on the kynurenines pathway but does not depend neither classical ways of quorum sensing or phnAB operon involved in the synthesis of anthranilate. However, the phenazines pathway could be involved in the production of this signal. By HPLC coupled with mass spectrometry, we were able to separate molecular species suppressing the TTSS and which are contained in this signal, but accurate identification requires further investigation. In a second time, we were interested to kynurenines produced by the bacterium. We confirmed that P. aeruginosa produces kynurenines and KynA is the key gene in the synthesis of these metabolites. We showed that tryptophan and kynurenine upregulate the production of kynurenines by acting on the expression of key genes. Other shares, we found that the bacterium modulates the activity of the kynurenines pathway depending on its state of growth. We showed that during the dialogue bacteria / host, the pathway of kynurenines in P. aeruginosa is stimulated by certain immune system components. With an acute lung infection model, we proved that kynurenines produced by the bacterium are important to its virulence. We hypothesized that the kynurenines could have an effect on the immune response, but this remains to be determined. In a third time, we focused on the protein OprF. We showed that mutation ΔoprF is causing an alteration in production but probably not the secretion of TTSS exotoxins. One known ligand of OprF is the gamma interferon. It modulates the pathway of kynurenines. OprF could therefore have a central role in various aspects of the regulation of virulence. So, we produced monoclonal anti-OprF which recognizes specifically the protein OprF. To verify the effectiveness of these antibodies, neutralization experiments of the bacteria in vitro and in vivo will be realized.

Les bactéries du genre Shigella sont responsables de la dysenterie bacillaire, ou shigellose, chez l'être humain, causant plus de 125 millions d'épisodes et 14 000 morts par an. Cette infection est caractérisée par l'inflammation et la destruction de la muqueuse intestinale. La bactérie utilise un système de sécrétion de type III (SST3) pour manipuler la physiologie des cellules épithéliales intestinales et du système immunitaire favorisant l'invasion de la muqueuse et enrayant la mise en place d'une réponse adaptative efficace. Le SST3 peut être comparé à une seringue moléculaire traversant la paroi bactérienne sous la forme d'anneaux membranaires, contenant une tige interne (MxiI), et d’une aiguille extracellulaire (MxiH). L'assemblage de cette dernière se termine par la mise en place d'un complexe d'extrémité formé par plusieurs copies des protéines IpaD et IpaB. Le SST3 prend en charge différentes classes de substrats à sa base via un complexe protéique comprenant l'ATPase Spa47. Les translocateurs (IpaB et IpaC) sont les premiers substrats à être sécrétés. Ceux-ci sont stockés dans le cytoplasme en complexe avec leur chaperon IpgC et sont recrutés à l'extrémité de l'aiguille lors du contact avec la membrane de la cellule hôte pour y former un pore à l'aide de la protéine IpaD. Ce pore permet l'injection des autres substrats du SST3 (effecteurs) qui vont interférer avec les voies de signalisation cellulaire. Il existe deux classes d'effecteurs, les effecteurs précoces (dont OspD1) stockés au préalable dans le cytoplasme et sécrétés suite au contact cellulaire. Ce contact active l’expression des effecteurs tardifs via un couplage assuré par deux complexes, OspD1-MxiE et translocateurs-IpgC. La sécrétion d’OspD1 et des translocateurs libère leurs partenaires qui agissent comme activateurs transcriptionnels. La régulation de la sécrétion dépend de plusieurs acteurs situés dans les différentes parties du SST3. Le complexe d'extrémité et la protéine MxiC contrôlent la sécrétion aux niveaux extra- et intracellulaires alors que l'aiguille transmettrait le signal de sécrétion entre ces deux complexes. Ce paradigme reste cependant encore peu compris et le mode de fonctionnement du complexe d’extrémité et de la protéine MxiC reste à éclaircir.<p>Nos travaux menés sur la protéine IpaD nous ont permis de mettre en évidence un phénotype de sécrétion intermédiaire. Celui-ci est caractérisé par la sécrétion des translocateurs et des effecteurs précoces, sans toutefois observer de sécrétion d’OspD1 et des effecteurs tardifs, suggérant un mécanisme de discrimination entre OspD1 et les effecteurs précoces. Ce phénotype de sécrétion est similaire à celui induit par l’interaction IpaD-désoxycholate. En effet, les variants d’ipaD restant fonctionnels pour la mise en place du pore provoquent également une augmentation de l’insertion des translocateurs et du pouvoir invasif. Nous avons également identifié la région d’IpaD nécessaire au maintien d’IpaB au niveau du complexe d’extrémité ainsi qu’un rôle de son domaine central dans l’insertion du pore. Nous avons enfin étudié l’effet d’anticorps monoclonaux anti-IpaD. Ces résultats nous ont permis de proposer un modèle de fonctionnement du complexe d’extrémité lors de l’insertion du pore, d’identifier les épitopes conférant une protection in vitro et in vivo ainsi que l’existence d’un polymorphisme qui empêche la liaison de ces anticorps à IpaD provenant d’autres sérotypes.<p>Notre étude sur MxiC a mis en évidence de nouveaux partenaires d’interaction (MxiI et IpgC). Ces résultats montrent que l’interaction MxiC-MxiI est nécessaire pour la régulation de la sécrétion des effecteurs précoces par MxiC. De même, la mutation mxiIQ67A provoque un phénotype similaire à la mutation mxiHK69A, ce qui suggère que le mécanisme de régulation impliquant l’aiguille est similaire pour la tige interne. Enfin, l’interaction renforcée MxiC-Spa47, via IpgC probablement couplée à un translocateur, apporte des pistes quant au rôle de MxiC dans la sécrétion des translocateurs.<p>Les rôles identifiés pour les différents régulateurs de la sécrétion ouvrent de nouvelles pistes pour la compréhension du fonctionnement du SST3. Leurs modes de fonctionnement restent cependant encore flous et nécessitent des études complémentaires.<p><p><p>Shigella are responsible for bacillary dysentery, or shigellosis, in human beings causing over 125 million episodes and 14 000 deaths per year. This infection is characterized by inflammation and destruction of the intestinal mucosa. The bacteria use a type III secretion system (T3SS) to manipulate the physiology of intestinal epithelial cells and the immune system favoring the invasion of the mucosa and halting the development of an efficient adaptive response. The T3SS can be compared to a molecular syringe that extends from the bacterial cell wall which contains an internal rod (MxiI), and an extracellular needle (MxiH). The assembly of the latter ends with assembly of a tip complex formed by multiple copies of IpaB and IpaD proteins. The T3SS recruits different classes of substrates at its base via a complex comprising the Spa47 ATPase. The translocators (IpaB and IpaC) are the first substrates to be secreted. They are stored in the cytoplasm in complex with their chaperone (IpgC) and are recruited at the needle tip upon contact with host cell membrane to form a pore via IpaD. This pore allows the injection of other substrates of the T3SS (effectors), which will interfere with the cellular signaling pathways. There are two classes of effectors, early effector (including OspD1) stored in the cytoplasm and secreted upon cell contact. This contact activates the expression of late effectors genes through a complex formed by MxiE (blocked by OspD1) and IpgC. Both proteins are released through OspD1 and translocators secretion. Secretion regulation depends on several actors located at different parts of the T3SS. The tip complex and the gatekeeper MxiC regulate secretion at the T3SS tip and base, the needle subunits transmitting a secretion signal between these two complexes. This paradigm, however, is still poorly understood and the operating mode of the tip complex and MxiC remains unclear.<p><p>Our work on IpaD protein allowed us to identify an intermediate secretion phenotype which is characterized by the secretion of translocators and early effector, but no secretion of OspD1 and late effectors, suggesting a discriminating mechanism between early effectors and OspD1. This secretion phenotype is similar to that induced by deoxycholate-IpaD interaction. Indeed, IpaD point mutants responsible for this phenotype cause an increase in the pore insertion and cell invasion. We also identified the region of IpaD necessary to maintain IpaB at the needle tip as well as a role of IpaD central domain in the pore insertion. We finally studied the effect of anti-IpaD monoclonal antibodies. These results allowed us to propose a working model of the tip complex end upon pore insertion, identify epitopes conferring protection in vitro and in vivo as well as the existence of a polymorphism that prevents the binding of these antibodies to IpaD from other serotypes.<p><p>Our MxiC study showed new interaction partners (MxiI and IpgC). These results showed that the MxiC-MxiI interaction is necessary for the regulation of early effectors secretion of by MxiC. Moreover, a mxiIQ67A mutation causes a phenotype similar to the mutation mxiHK69A, suggesting that the regulatory mechanism involving the needle is shared by the inner rod. Finally, the enhanced interaction MxiC-Spa47 through IpgC, probably in complex with a translocator, provides clues for the role of MxiC in translocators secretion.<p><p>The roles identified for the various regulators of secretion open up new avenues for understanding how the T3SS functions. Their ways of working are however still unclear and require further study.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

A Transferência Horizontal de Genes (THG) é um dos principais mecanismos de evolução bacterianos, impactando a evolução de praticamente todas famílias gênicas. Neste trabalho identificamos e avaliamos padrões de possíveis transferências horizontais de genes pertencentes a duas classes funcionais de dois níveis taxonômicos distintos. Caracterizamos a ocorrência e evolução de 45 genes importantes para a fixação de N2 em 479 genomas de Proteobacteria. Identificamos cinco potenciais aquisições de genes ligados a fixação de N2 por linhagens de Proteobacteria, as quais foram identificadas consistentemente em 36 dos genes analisados. Realizamos predições de transferências horizontais dos 45 entre todos os 479 genomas de Proteobacteria e identificamos possíveis enriquecimentos de THG, provavelmente ligados à sinais filogenéticos e ecológicos. Desenvolvemos um pipeline para identificação semi-automática de efetores do Sistema Secretor do Tipo III em Aeromonas, o qual reportou 21 famílias de potenciais efetores presentes em 105 genomas. Entre os 21 efetores identificados 17 foram descritos pela 1º vez em Aeromonas, corroborando a sensibilidade de nosso pipeline. Com o auxílio de nossos colaboradores foram realizados testes de citotoxidade para efetores identificados in silico, e apenas quatro não inibiram o crescimento de Saccharomyces cerevisiae. Por fim, desenvolvemos um método para agrupamento de famílias gênicas com histórias evolutivas similares que não requer a reconstrução de árvores filogenéticas, aumentando a eficiência computacional. Aplicamos o método desenvolvido para reconstrução da filogenia de Aeromonas, o qual mostrou-se compatível com dados presentes na literatura. / Horizontal Gene Transfer (HGT) is one of main mechanisms of bacterial evolution, affecting virtually all gene families. In this document we identified and assessed putative horizontal transfers of genes from two functional classes from two distinct taxonomic levels. We characterized the distribution and evolution of 45 genes important to N2 fixation among 479 Proteobacteria genomes. We identified five potential distinct acquisitions of such genes by Proteobacteria lineages. The distinct origins are consistently identified in 36 out of the 45 assessed genes. We computed possible horizontal transfers of the 45 genes among the 479 Proteobacteria genomes, and we identified enrichments of HGT, likely related to phylogenetic and ecological signals. We developed a semi-automated pipeline to identify effectors of the Type III Secretion System within Aeromonas, which reported 21 putative effector families distributed among 105 genomes. Among the 21 likely effectors 17 have been described in Aeromonas for the first time, highlighting the sensibility of our pipeline. Our colaborators performed cytotoxicity tests for the 21 likely effector families identified by in silico analysis, and only four did not inhibited Saccharomyces cerevisiae growth. Lastly, we developed a method to cluster gene families according to shared evolutionary history, without the requirement of phylogenetic tree reconstruction, increasing computational efficiency. We applied this proposed method during Aeromonas phylogenetic reconstruction, and it showed up compatible with data available on the literature.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

<p>The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.</p>

The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.