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Molecular Characterisation of Salmonella enterica Serovar Sofia in AustraliaGan Teck Fong, Emily, xf_dksfwm@yahoo.com January 2008 (has links)
Despite its high isolation frequency in Australian chickens, S. II Sofia is rarely associated with animals or human salmonellosis as this serovar is avirulent in nature. The reason for its persistence and avirulence is unknown as very few studies have been conducted on the epidemiology and pathogenicity of this strain. This study details the various experimental methods utilised to investigate the genetic relatedness and molecular mechanisms involved in S. II Sofia pathogenesis. Using PFGE and Rep-PCR, the Australian S. II Sofia isolates were found to show limited genetic diversity and probably share a clonal relationship. A majority of the S. II Sofia isolates were not geographically restricted with the predominant pattern subtype spread out among the isolates from various states. Distribution and variation of the SPI-associated virulence genes within S. II Sofia was also examined. Based on RFLP and sequence analysis, most of the differences observed in SPI1 to SPI5 of S. II Sofia could be attributed to a loss or gain of restriction cleavage sites within these regions. However, a number of genes in SPI1, SPI2, SPI3 and SPI5 were found to have accumulated changes (mutations, insertions and deletions) that could have affected gene transcription and/or protein translation - these genes have been shown to be involved in different aspects of the virulence process. The avirulence of S. II Sofia is probably not the result of a single genetic change but rather a series of alterations to a large number of its virulence-associated genes. Plasmid-mediated virulence was also assessed in S. II Sofia isolates. Southern hybridisation with probes derived from the virulence plasmid of S. Typhimurium indicated either the total absence of the virulence plasmid or possible presence of a virulence plasmid containing major deletions. Clones were constructed with the missing spv operon using high-copy pCR®2.1 and low-copy pWSK29 plasmids and the adherence, invasion and intracellular survival of the mutant strain was evaluated in vitro. The presence of spvRABCD was shown to have no effect on intracellular survival and replication. Although the cloning of spv with pCR®2.1 was observed to significantly increase invasiveness of S. II Sofia, it was not capable of restoring the invasive ability of S. II Sofia to the level of pathogenic S. Typhimurium 82/6915. On the other hand, the uneven adherence and invasion ability of the other mutant strains appeared to be linked to the presence of pWSK29 and this observation is further supported by RT-PCR analysis of the clones - indicating that perhaps pWSK29 is not a suitable vector for this study. Wild-type S. II Sofia isolates are unlikely to regain full pathogenicity because of the numerous mutations in many important virulence genes: even the chance acquisition of a virulence factor (e.g. spvRABCD) is not sufficient to completely restore S. II Sofia virulence. Therefore, S. II Sofia should not be considered similar to other Salmonella spp. when monitoring Salmonellae in food samples.
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Modulating the gut microbiota with a synthetic stool “MET-1”: protective effects in animal models of antibiotic associated colitisMartz, SARAH-LYNN 02 October 2013 (has links)
Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-29 21:18:18.966
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Study of the dissemination of cefoxitin-resistant Salmonella enterica serovar Heidelberg from human, abattoir poultry and retail poultry sourcesEdirmanasinghe, Romaine Cathy Shalini 15 September 2016 (has links)
This study characterized Salmonella enterica serovar Heidelberg from human, abattoir poultry and retail poultry isolates to examine the molecular relationships of cefoxitin resistance between these groups. A total of 147 S. Heidelberg (70 cefoxitin-resistant and 77 cefoxitin-susceptible) isolates were studied. All cefoxitin-resistant isolates were also resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur and ceftriaxone, and all contained the CMY-2 gene. Pulsed-field gel electrophoresis typing illustrated that 93.9% isolates clustered together with ≥ 90% similarity. Core genome analysis using whole genome sequencing identified 12 clusters of isolates with zero to four single nucleotide variations. These clusters consisted of cefoxitin-resistant and susceptible human, abattoir poultry and retail poultry isolates. Analysis of CMY-2 plasmids from cefoxitin-resistant isolates revealed all belonged to incompatibility group I1. Analysis of plasmid sequences using WGS revealed high identity (95-99%) to a previously described plasmid (pCVM29188_101) found in Salmonella Kentucky. When compared to pCVM29188_101, all sequenced cefoxitin-resistant isolates were found to carry one of ten possible variant plasmids. The discovery of several clusters of isolates from different sources with zero to four SNVs suggests that transmission between human, abattoir poultry and retail poultry sources may be occurring. The classification of newly sequenced plasmids into one of ten sequence variant types suggests transmission of a common CMY-2 plasmid amongst S. Heidelberg with variable genetic backgrounds. / October 2016
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Characterization of <i>Salmonella</i> Bacteriophages Isolated from Farm Environments for Use in Decontamination of Liquid Whole EggYi, Yue January 2019 (has links)
No description available.
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Salmonella enterica serovar enteritidis requires the type three secretion system-1/2 to invade/survive in chicken oviduct epithelial cells and to modulate innate immune responsesLi, Shuhui 03 May 2008 (has links)
Contaminated poultry and egg products are major sources of Salmonella enterica serovar Enteritidis (S. enteritidis, SE) infections in humans. Colonization of SE in chicken reproductive tract results in the production of contaminated commercial shell-eggs and fertilized hatchery eggs. The complex pathogen-host interactions during SE colonization of chicken reproductive tract are largely unknown. This study was aimed at determining the pathogenic roles of the type three secretion systems (TTSS-1 and TTSS-2) in SE infection of chicken oviduct epithelial cells (COEC). A series of SE strains carrying mutations in the genes encoding structure or effector proteins of TTSS-1 and TTSS-2 were constructed. The invasiveness and intracellular survival rate of each SE strain as well as the host innate immune responses induced by the infections were evaluated. The results demonstrate that both TTSS-1 and TTSS-2 are required by SE to invade COEC which involve genes encoding effector proteins SipA, SopB, SopE2, and PipB. In addition to their involvement in host cell invasion, sipA and sipB are also necessary for the survival or replication of SE inside COEC. Inactivation of TTSS-2 genes (ssaV and pipB) resulted in an enhanced bacterial proliferation inside COEC. The data from this study also show that SE infection triggers pro-inflammatory responses in COEC and TTSS-1 is involved in the expression of iNOS and IL-8, a CXC chemokine. TTSS-1 and TTSS-2 are not necessary for induction of K203, MIP-1β, and IL-10 or suppression of TGF-β3 in COEC.
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Effect of Morphine on Immune Responses and InfectionBreslow, Jessica January 2010 (has links)
Opioids have been shown to modulate immune function in a variety of assays and animal models. In a more limited number of studies, opioids have been shown to sensitize to infection. Heroin, the prototypical opioid drug of abuse, is rapidly metabolized to morphine in the body. Morphine has been used as an analgesic for hundreds of years, and continues to be a drug of choice for treating pain in ICU and trauma patients. The continued use of these opioid compounds in humans warrants further investigation of their effect on immune responses against, and progression of, common bacterial infections. Two infections were investigated in this thesis using murine models, Acinetobacter baumannii and Salmonella typhimurium. A recent increase in the prevalence of A. baumannii infections among healthy, but wounded, military personnel, lead to the hypothesis that analgesic morphine might sensitize to infection with this multiply-drug resistant bacterium. A systemic, intraperitoneal A. baumannii infection model was established in mice that resulted in rapid, disseminated disease where animals became septic as organisms replicated in the blood, lungs, and other organs. This model was used to investigate the role of various parameters of innate immune defenses to Acinetobacter. Neutralization of neutrophils by antibody depletion greatly sensitized to this infection. Infection resulted in a rapid, biphasic induction of both IL-17 and the chemokine, KC/CXCL1, a major chemotactic factor for neutrophils, that continued to rise through 18h after bacterial inoculation. However, depletion of either IL-17 or KC/CXCL1 using monoclonal antibodies failed to sensitize to Acinetobacter infection. Further, IL-17 receptor KO mice were not sensitized to this infection. Collectively, these results suggest that there must be other chemotactic factors for neutrophils that can compensate for the absence of IL-17 and KC. Morphine, delivered by extended release pellet, sensitized two strains of mice to two strains of Acinetobacter, as measured by mortality to a sublethal challenge dose, and this effect was blocked by administration of the opioid-receptor antagonist, naltrexone. . Morphine increased Acinetobacter burdens in the organs and blood of infected mice, and increased the levels of pro-inflammatory cytokines. Evidence for an effect of morphine on neutrophil infiltration was obtained. Morphine decreased the total numbers of cells, as well as the total numbers of neutrophils and macrophages infiltrating into the peritoneal cavity. This inhibition of neutrophil accumulation correlated with suppression of levels of both IL-17 and KC/CXCL1. The evidence supports the conclusion that morphine sensitizes to Acinetobacter infection by suppressing the response of neutrophils, potentially via depression of neutrophil chemotactic factors IL-17 and KC. However, taken together with the data above there are probably additional factors in addition to IL-17 and KC that are sensitizing the animals to infection in the presence of morphine. In addition to these studies, the opioid-receptor dependency of morphine-mediated sensitization to Salmonella enteric serovar Typhimurium was examined. Previous experiments had determined that extended release morphine pellets sensitized mice to a sublethal dose of Salmonella, as determined by survival and bacterial burdens in the organs of infected mice, but naltrexone resulted in only incomplete reversal of the morphine-mediated effects. To further characterize the receptor dependency of the observed phenomenon, mu-opioid receptor knockout (MORKO) mice were used. MORKO mice were found to be completely resistant to the lethal effects of morphine plus infection observed in wild-type (WT) mice. In addition, MORKO mice showed greatly reduced bacterial burdens and pro-inflammatory cytokine levels when treated with morphine and challenged with a sublethal challenge dose of Salmonella, in comparison to WT mice. In summary, the studies presented in this thesis explored basic mechanisms of innate immunity to A. baumannii using a systemic model of infection. The work provides additional evidence that morphine sensitizes to infection, using models of Acinetobacter and Salmonella in mice. An implication of this work is use of caution in the administration of opioids in patients that are susceptible to opportunistic infections. / Microbiology and Immunology
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Étude fonctionnelle de l’opéron fimbriaire stg de Salmonella enterica sérovar TyphiForest, Chantal 11 1900 (has links)
La bactérie Salmonella enterica sérovar Typhi (S. Typhi) provoque la fièvre typhoïde chez les humains et constitue un problème de santé publique important. La majorité de nos connaissances sur la pathogenèse de cette bactérie provient du modèle de fièvre entérique chez la souris causée par le sérovar Typhimurium. Peu d’études se sont penchées sur les facteurs de virulence uniques au sérovar Typhi, ni sur la possibilité que les pseudogènes retrouvés dans son génome puissent être fonctionnels. Le fimbria stg, unique au sérovar Typhi, renferme un codon d’arrêt TAA prématuré dans le gène stgC qui code pour le placier responsable de l’assemblage des sous-unités fimbriaires à la surface de la bactérie. Ainsi, le fimbria stg a été classifié dans la liste des pseudogènes non-fonctionnels. Les objectifs de cette étude étaient d’évaluer l’implication du fimbria stg lors de l’interaction avec les cellules humaines, puis de vérifier l’importance du pseudogène stgC lors de la biogenèse fimbriaire.
Dans une première partie, la transcription de stg a été évaluée à l’aide d’une fusion lacZ. Malgré des niveaux d’expression observés généralement faibles en milieu riche, la croissance en milieu minimal a favorisé la transcription de l’opéron. La délétion complète de l’opéron fimbriaire stgABCD du génome de S. Typhi a été réalisée par échange allélique, puis a été complémentée sur un plasmide. Il a été démontré que la présence de stg chez S. Typhi, S. Typhimurium et E. coli contribue à une adhérence accrue sur les cellules épithéliales humaines. De plus, ce fimbria semble agir comme une structure anti-phagocytaire lors de l’interaction avec des macrophages humains. Ainsi, l’opéron stg semble fonctionnel, malgré son codon d’arrêt prématuré, puisque des phénotypes ont été observés.
La seconde partie de cette étude consistait à vérifier le rôle joué par le pseudogène stgC dans la biogenèse du fimbria. Différentes variantes de l’opéron ont été générées, clonées dans un vecteur inductible à l’arabinose, puis transformées dans la souche afimbriaire d’E. coli ORN172. La translocation de la sous-unité fimbriaire StgD à la surface de la bactérie a été évaluée chez ces différents mutants par immunobuvardage de type Western. Cette expérience a permis de démontrer que le pseudogène stgC est essentiel pour l’exportation de la sous-unité StgD à la surface. L’ajout d’une étiquette de 6-histidines en C-terminal de StgC a permis de confirmer la traduction complète du gène, malgré le codon d’arrêt TAA prématuré. Le séquençage peptidique a révélé l’insertion d’une tyrosine à ce codon. Une fusion traductionnelle avec la protéine verte fluorescente a révélé qu’environ 0.8% de l’ARNm peut être traduit et permet la production complète du placier.
Ce projet a permis la caractérisation d’un facteur de virulence unique à S. Typhi et constitue une étape de plus vers la compréhension de ses mécanismes de pathogenèse. Il s’agit de la première démonstration chez les bactéries de la fonctionnalité d’un gène interrompu prématurément par un codon d’arrêt TAA. / Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans and is considered as an important health problem. Most of our knowledge on the pathogenesis of this bacterium comes from an enteric fever model in mice caused by serovar Typhimurium. Few studies have examined the virulence factors unique to serovar Typhi or the possibility that pseudogenes harbored in its genome may be functional. stg fimbriae are found only within the serovar Typhi genome and contain a premature TAA stop codon in the stgC gene encoding the usher responsible for the assembly of fimbrial subunits at the bacterial surface. Thus, the stg fimbria has been classified among the list of non-functional pseudogenes. The objectives of this study were to assess the involvement of stg fimbriae during interaction with human cells, and then to evaluate the importance of the stgC pseudogene in fimbrial biogenesis.
First, stg transcription was evaluated using a lacZ fusion. Despite low expression levels generally observed in rich medium, growth in minimal medium promoted transcription of the operon. Complete deletion of the stgABCD fimbrial operon from S. Typhi was performed by allelic exchange and was complemented on a plasmid. It has been shown that the presence of stg in S. Typhi, S. Typhimurium and E. coli contributes to increased adherence to human epithelial cells. In addition, the fimbriae seem to act as an anti-phagocytic structure during the interaction with macrophages. Thus, the stg operon appears to be functional despite its premature codon, as phenotypes were observed.
The second part of this study involved testing the role of the stgC pseudogene in fimbrial biogenesis. Different variants of the operon were generated, cloned into an arabinose inducible vector, and then transformed into afimbriated E. coli strain ORN172. Translocation of the StgD subunit to the cell surface of the different mutants was evaluated using Western blot. This experiment demonstrated that stgC is essential for export of the StgD subunit to the cell surface. The addition of a 6-histidine tag at the C-terminal end of StgC confirmed the complete translation of the gene, despite the premature TAA stop codon. Peptide sequencing revealed the insertion of a tyrosine at this codon. A translational fusion with the green fluorescent protein demonstrated that approximately 0.8% of the mRNA can be translated to allow full production of the usher.
This project allowed characterization of a virulence factor unique to S. Typhi and is a step closer towards better understanding of its pathogenesis mechanisms. This is the first demonstration in bacteria of the functionality of a gene which is interrupted by a premature TAA stop codon.
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Caractérisation et délétion de tous les systèmes d'adhésion connus de Salmonella enterica sérovar TyphiDavid, Élise 08 1900 (has links)
Les fimbriae sont des structures protéiques extracellulaires retrouvées chez une vaste diversité de bactéries. Ces structures ont fait l’objet de nombreuses études et sont maintenant reconnus pour leur implication dans l’adhésion et l’invasion aux cellules eucaryotes, mais aussi dans la production de biofilms. Ils sont groupés selon leur voie de sécrétion. Certains utilisent une machinerie spécifique et individuelle, c’est le cas des pili de type IV, tandis que d’autres utilisent la voie de sécrétion générale suivit d’une voie spécifique telle que la voie du chaperon-placier (« Chaperon Usher Pathway ») (fimbriae CUP) ou la voie de nucléation précipitation (« nucleation precipitation pathway ») (Curli). Malgré toutes les connaissances actuelles concernant les fimbriae, très peu d’informations sont disponibles quant aux fimbriae de Salmonella enterica sérovar Typhi (S. Typhi). Ce pathogène unique à l’homme est l’agent étiologique de la fièvre typhoïde. Puisque les fimbriae sont reconnus pour être impliqués dans l’adaptation à l’hôte, nous avons décidé d’étudier davantage l’arsenal fimbriaire de S. Typhi, dans l’espoir d’identifier des facteurs de virulence uniques à S. Typhi et impliqués dans la ségrégation de l’hôte. La souche S. Typhi ISP1820 possède 14 opérons codant pour des systèmes d’adhésion, mais plusieurs contiennent des pseudogènes et leur expression n’a jamais été observée in vitro. Afin d’étudier les systèmes d’adhésion de S. Typhi, nous avons supprimé chaque opéron du génome individuellement et cumulativement à l’aide une technique de mutagénèse par échange allélique. Ainsi, nous avons testé chaque mutant individuel et la souche mutante pour tous les systèmes d’adhésion dans plusieurs essais tels que des infections de cellules épithéliales et de macrophages, de mobilité et de formation de biofilm. Nous avons aussi évalué l’expression des fimbriae lors de différentes conditions de croissance en laboratoire par RT-PCR. Tous les tests réalisés nous ont permis de découvrir que plusieurs opérons fimbriaires de S. Typhi sont opérationnels et utilisés pour différentes fonctions par la bactérie. / Fimbriae are extracellular proteinaceous appendages found in many bacteria. They are widely studied and believe to be implicated in several cellular functions such as adhesion, invasion of eukaryotic cells, and biofilm production. They are classified depending on their pathway of secretion: some, like type IV pili, use self-specific machinery, while others use the general secretory pathway followed by their own assembly pathway such as the Chaperon Usher Pathway (CUP fimbriae) and the nucleation precipitation pathway (curli). Despite everything that is known about these structures, little has been discovered regarding fimbrial systems of Salmonella enterica serovar Typhi (S. Typhi). This pathogen is a human restricted serovar and the etiological agent of typhoid fever. Since fimbriae have been implicated in host adaptation, we have decided to further study S. Typhi fimbrial arsenal in the hope of uncovering virulence factors unique to S. Typhi and implicated in host specificity. The S. Typhi ISP1820 strain carries 14 operons encoding for fimbrial structures, but many are believed pseudogenes or are not expressed in vitro. In order to study these different adhesion systems in S. Typhi, we have deleted each one individually and cumulatively by allelic exchange mutagenesis. Hence, we have tested every individual mutation and the mutant strain deprived of all 14 operons in many different assays including epithelial cell and macrophage infection, mobility, and biofilm formation. We also evaluate expression during growth under laboratory conditions by RT-PCR. These experiments have allowed us to discover that many of S. Typhi fimbriae are functional, expressed, and used by the bacteria in many different processes.
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Caractérisation et délétion de tous les systèmes d'adhésion connus de Salmonella enterica sérovar TyphiDavid, Élise 08 1900 (has links)
Les fimbriae sont des structures protéiques extracellulaires retrouvées chez une vaste diversité de bactéries. Ces structures ont fait l’objet de nombreuses études et sont maintenant reconnus pour leur implication dans l’adhésion et l’invasion aux cellules eucaryotes, mais aussi dans la production de biofilms. Ils sont groupés selon leur voie de sécrétion. Certains utilisent une machinerie spécifique et individuelle, c’est le cas des pili de type IV, tandis que d’autres utilisent la voie de sécrétion générale suivit d’une voie spécifique telle que la voie du chaperon-placier (« Chaperon Usher Pathway ») (fimbriae CUP) ou la voie de nucléation précipitation (« nucleation precipitation pathway ») (Curli). Malgré toutes les connaissances actuelles concernant les fimbriae, très peu d’informations sont disponibles quant aux fimbriae de Salmonella enterica sérovar Typhi (S. Typhi). Ce pathogène unique à l’homme est l’agent étiologique de la fièvre typhoïde. Puisque les fimbriae sont reconnus pour être impliqués dans l’adaptation à l’hôte, nous avons décidé d’étudier davantage l’arsenal fimbriaire de S. Typhi, dans l’espoir d’identifier des facteurs de virulence uniques à S. Typhi et impliqués dans la ségrégation de l’hôte. La souche S. Typhi ISP1820 possède 14 opérons codant pour des systèmes d’adhésion, mais plusieurs contiennent des pseudogènes et leur expression n’a jamais été observée in vitro. Afin d’étudier les systèmes d’adhésion de S. Typhi, nous avons supprimé chaque opéron du génome individuellement et cumulativement à l’aide une technique de mutagénèse par échange allélique. Ainsi, nous avons testé chaque mutant individuel et la souche mutante pour tous les systèmes d’adhésion dans plusieurs essais tels que des infections de cellules épithéliales et de macrophages, de mobilité et de formation de biofilm. Nous avons aussi évalué l’expression des fimbriae lors de différentes conditions de croissance en laboratoire par RT-PCR. Tous les tests réalisés nous ont permis de découvrir que plusieurs opérons fimbriaires de S. Typhi sont opérationnels et utilisés pour différentes fonctions par la bactérie. / Fimbriae are extracellular proteinaceous appendages found in many bacteria. They are widely studied and believe to be implicated in several cellular functions such as adhesion, invasion of eukaryotic cells, and biofilm production. They are classified depending on their pathway of secretion: some, like type IV pili, use self-specific machinery, while others use the general secretory pathway followed by their own assembly pathway such as the Chaperon Usher Pathway (CUP fimbriae) and the nucleation precipitation pathway (curli). Despite everything that is known about these structures, little has been discovered regarding fimbrial systems of Salmonella enterica serovar Typhi (S. Typhi). This pathogen is a human restricted serovar and the etiological agent of typhoid fever. Since fimbriae have been implicated in host adaptation, we have decided to further study S. Typhi fimbrial arsenal in the hope of uncovering virulence factors unique to S. Typhi and implicated in host specificity. The S. Typhi ISP1820 strain carries 14 operons encoding for fimbrial structures, but many are believed pseudogenes or are not expressed in vitro. In order to study these different adhesion systems in S. Typhi, we have deleted each one individually and cumulatively by allelic exchange mutagenesis. Hence, we have tested every individual mutation and the mutant strain deprived of all 14 operons in many different assays including epithelial cell and macrophage infection, mobility, and biofilm formation. We also evaluate expression during growth under laboratory conditions by RT-PCR. These experiments have allowed us to discover that many of S. Typhi fimbriae are functional, expressed, and used by the bacteria in many different processes.
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Étude fonctionnelle de l’opéron fimbriaire stg de Salmonella enterica sérovar TyphiForest, Chantal 11 1900 (has links)
La bactérie Salmonella enterica sérovar Typhi (S. Typhi) provoque la fièvre typhoïde chez les humains et constitue un problème de santé publique important. La majorité de nos connaissances sur la pathogenèse de cette bactérie provient du modèle de fièvre entérique chez la souris causée par le sérovar Typhimurium. Peu d’études se sont penchées sur les facteurs de virulence uniques au sérovar Typhi, ni sur la possibilité que les pseudogènes retrouvés dans son génome puissent être fonctionnels. Le fimbria stg, unique au sérovar Typhi, renferme un codon d’arrêt TAA prématuré dans le gène stgC qui code pour le placier responsable de l’assemblage des sous-unités fimbriaires à la surface de la bactérie. Ainsi, le fimbria stg a été classifié dans la liste des pseudogènes non-fonctionnels. Les objectifs de cette étude étaient d’évaluer l’implication du fimbria stg lors de l’interaction avec les cellules humaines, puis de vérifier l’importance du pseudogène stgC lors de la biogenèse fimbriaire.
Dans une première partie, la transcription de stg a été évaluée à l’aide d’une fusion lacZ. Malgré des niveaux d’expression observés généralement faibles en milieu riche, la croissance en milieu minimal a favorisé la transcription de l’opéron. La délétion complète de l’opéron fimbriaire stgABCD du génome de S. Typhi a été réalisée par échange allélique, puis a été complémentée sur un plasmide. Il a été démontré que la présence de stg chez S. Typhi, S. Typhimurium et E. coli contribue à une adhérence accrue sur les cellules épithéliales humaines. De plus, ce fimbria semble agir comme une structure anti-phagocytaire lors de l’interaction avec des macrophages humains. Ainsi, l’opéron stg semble fonctionnel, malgré son codon d’arrêt prématuré, puisque des phénotypes ont été observés.
La seconde partie de cette étude consistait à vérifier le rôle joué par le pseudogène stgC dans la biogenèse du fimbria. Différentes variantes de l’opéron ont été générées, clonées dans un vecteur inductible à l’arabinose, puis transformées dans la souche afimbriaire d’E. coli ORN172. La translocation de la sous-unité fimbriaire StgD à la surface de la bactérie a été évaluée chez ces différents mutants par immunobuvardage de type Western. Cette expérience a permis de démontrer que le pseudogène stgC est essentiel pour l’exportation de la sous-unité StgD à la surface. L’ajout d’une étiquette de 6-histidines en C-terminal de StgC a permis de confirmer la traduction complète du gène, malgré le codon d’arrêt TAA prématuré. Le séquençage peptidique a révélé l’insertion d’une tyrosine à ce codon. Une fusion traductionnelle avec la protéine verte fluorescente a révélé qu’environ 0.8% de l’ARNm peut être traduit et permet la production complète du placier.
Ce projet a permis la caractérisation d’un facteur de virulence unique à S. Typhi et constitue une étape de plus vers la compréhension de ses mécanismes de pathogenèse. Il s’agit de la première démonstration chez les bactéries de la fonctionnalité d’un gène interrompu prématurément par un codon d’arrêt TAA. / Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans and is considered as an important health problem. Most of our knowledge on the pathogenesis of this bacterium comes from an enteric fever model in mice caused by serovar Typhimurium. Few studies have examined the virulence factors unique to serovar Typhi or the possibility that pseudogenes harbored in its genome may be functional. stg fimbriae are found only within the serovar Typhi genome and contain a premature TAA stop codon in the stgC gene encoding the usher responsible for the assembly of fimbrial subunits at the bacterial surface. Thus, the stg fimbria has been classified among the list of non-functional pseudogenes. The objectives of this study were to assess the involvement of stg fimbriae during interaction with human cells, and then to evaluate the importance of the stgC pseudogene in fimbrial biogenesis.
First, stg transcription was evaluated using a lacZ fusion. Despite low expression levels generally observed in rich medium, growth in minimal medium promoted transcription of the operon. Complete deletion of the stgABCD fimbrial operon from S. Typhi was performed by allelic exchange and was complemented on a plasmid. It has been shown that the presence of stg in S. Typhi, S. Typhimurium and E. coli contributes to increased adherence to human epithelial cells. In addition, the fimbriae seem to act as an anti-phagocytic structure during the interaction with macrophages. Thus, the stg operon appears to be functional despite its premature codon, as phenotypes were observed.
The second part of this study involved testing the role of the stgC pseudogene in fimbrial biogenesis. Different variants of the operon were generated, cloned into an arabinose inducible vector, and then transformed into afimbriated E. coli strain ORN172. Translocation of the StgD subunit to the cell surface of the different mutants was evaluated using Western blot. This experiment demonstrated that stgC is essential for export of the StgD subunit to the cell surface. The addition of a 6-histidine tag at the C-terminal end of StgC confirmed the complete translation of the gene, despite the premature TAA stop codon. Peptide sequencing revealed the insertion of a tyrosine at this codon. A translational fusion with the green fluorescent protein demonstrated that approximately 0.8% of the mRNA can be translated to allow full production of the usher.
This project allowed characterization of a virulence factor unique to S. Typhi and is a step closer towards better understanding of its pathogenesis mechanisms. This is the first demonstration in bacteria of the functionality of a gene which is interrupted by a premature TAA stop codon.
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