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.

No description available.

Au Vietnam, les informations sur la contamination de la viande de volaille par les salmonelles sont presque limitées. L’étude cherche à comparer la prévalence des salmonelles entre les marchés traditionnels et les supermarchés ainsi qu’entre les carcasses fraîches et congelées en plus de mesurer la température interne au moment de l’achat. Deux cent quarante-cinq carcasses de poulets entiers ont été achetées des marchés et des supermarchés dans sept arrondissements de la ville de Hanoi au Vietnam de juin à juillet 2011. L’échantillonnage a inclu 110 carcasses fraîches de marchés traditionnels (F/M), 109 carcasses fraîches des supermarchés (F/SM) et 26 carcasses congelées des supermarchés (FZ/SM). La température intérieure des carcasses a été évalué au moment de l’achat des carcasses. Salmonella a été isolé à partir de rinçage de carcasses et les isolats ont été sérotypés. La prévalence de carcasses positives pour Salmonella était de 66,5% (163/245) et variait entre les trois catégories : 84,55% (93/110) de F/M, 59,63% (65/109) de F/SM et 19,23% (5/26) de FZ/SM (P<0.05). Pour un total de 25 sérovars détectés, le sérovar principal fut Agona (24,78%) suivi de Albany (20,43%) et enfin Corvallis (10%). Deux des sérovars repérés se retrouvaient sur les mêmes carcasses pour 66 échantillons (26,9%). La température interne des carcasses des marchés traditionnels et des supermarchés était associé une différence significative (P < 0.05) avec une température moyenne de 27,3°C et 15,8°C respectivement. Cette étude dévoile une prévalence élevée de Salmonellaspp.des carcasses de poulets à Hanoi et démontre une difficulté partagée par tous les types de marchés à maintenir une température adéquate des carcasses. / In Vietnam, the data on the prevalence of Salmonella contamination in retail chicken meat is limited. We wanted to compare that prevalence at traditional and modern supermarkets, as well as in fresh versus frozen carcasses, and to verify the inner carcass temperatures at time of purchase. A collection of 245 whole chicken carcasses were purchased from traditional markets and supermarkets, in seven urban district areas of Hanoi in June and July, 2011. Sampling plan included 110 fresh chickens from traditional markets (F/M), 109 fresh chickens from supermarkets (F/SM) and 26 frozen chickens from supermarkets (FZ/SM). The inner carcass temperature was measured at the time of purchase. Salmonella was isolated from carcass rinses and isolates were serotyped. The overall prevalence of Salmonella-positive carcasses was 66.5% (163/245). The Salmonella prevalence in the three types of chickens varied significantly, 84.55% (93/110) from F/M, 59.63% (65/109) from F/SM and 19.23% (5/26) from FZ/SM (P< 0.05). A total of 25 serovars were recovered. The predominant serovars were Agona (24.78%), Albany (20.43%) and Corvallis (10%). Two different serovars were isolated and coexisted on the same carcass in 66 samples (26.9%). The inner carcass temperatures of fresh samples from traditional markets and supermarkets were significantly different (P <0.05) with a mean inner carcass temperature of 27.3oC and 15.8oC respectively. This study revealed a high prevalence of Salmonella sp. from retail chickens in Hanoi and uncovered the difficulty encountered by all market types to store broiler chicken carcasses at a safe temperature.

Salmonella enterica sérovar Typhi (S. Typhi) est l’agent responsable de la fièvre typhoïde et cause environ 200 000 morts et 27 millions de cas annuellement. C’est un pathogène entérique dont le réservoir est restreint à l’Homme. Les raisons de cette restriction d’hôte sont méconnues et pourraient dépendre de l’expression de facteurs d’adhésion à des étapes importantes au cours de la pathogenèse. L’annotation bioinformatique du génome de S. Typhi identifie 12 fimbriae de type chaperon-placier (FCP), un curli ainsi qu’un pilus de type IV. L’objectif de ce projet de recherche est d’étudier ces systèmes d’adhésion peu caractérisés. D’abord, le niveau d’expression de ces gènes a été évalué dans différentes conditions de culture in vitro en utilisant une approche de gènes rapporteurs. L’expression des 14 systèmes d’adhésion a été détectée. Nos résultats indiquent qu’une carence en fer favorise l’expression des opérons bcf et csg. Indépendamment du fer, l’expression de bcf, csg, pil, sef, sta, stc, stg et sth est influencée par la richesse nutritive du milieu. L’incubation en milieu LB liquide favorise l’expression de la plupart des systèmes d’adhésion par rapport à un milieu LB liquide sans agitation ou un milieu LB solide. En somme, l’expression des systèmes d’adhésion de S. Typhi a été observée et est influencée par des conditions environnementales. Dans un second volet, nous avons tent de surexprimer les différents systèmes d’adhésion chez une souche d’E. coli ou de S. Typhi afimbriaire. Avec cette approche, nous avons été en mesure de démontrer que l’opéron tcf encode pour un fimbria fonctionnel que l’on a pu observer en microscopie électronique. L’expression de tcf chez une souche afimbriaire d’E. coli et S. Typhi a également diminué leur capacité d’adhésion à des cellules épithéliales intestinales humaines lors d’essais in vitro. Nos observations démontrent que l’expression des systèmes d’adhésion retrouvés chez S. Typhi est influencée par les conditions enviroi9onnementales. Au moins un de ces systèmes est fonctionnel. Ceci suggère une contribution des systèmes d’adhésion retrouvés chez S. Typhi lors de l’interaction de ce pathogène avec l’humain. / Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of typhoid fever which causes more than 200 000 deaths and 27 million cases worldwide, mostly in south Asia. This pathogen can only cause significant symptoms in humans, which are the only recognized animal reservoir. This host restriction is not clearly understood and could depend on the expression of adhesion systems during critical pathogenesis steps. Bioinformatic studies on S. Typhi predict 12 chaperone-usher fimbriae, one curli and one type IV secretion system. The aim of the project was to study those poorly described adhesion systems using two different methodologies. First, transcription levels were evaluated in different in vitro growth conditions using both gfp and β-galactosidase reporter genes. The expression of the 14 adhesion systems was detected, even if some of them were poorly expressed. The expression of bcf and csg was higher during iron-deficiency. Also, the availability of nutrients had an impact on bcf, csg, pil, sef, sta, stc, stg and sth expression, independently of the presence of iron. Most of the adhesion systems showed higher expression levels in liquid LB media with aeration compared to the same media without aeration or supplemented with agar. Secondly, several S. Typhi adhesion systems were cloned into an inducible expression plasmid introduced in both an afimbriated E. coli K-12 strain (ORN172) and an afimbriated S. Typhi strain (ISP1820). This approach enabled us to directly observe the presence of tcf by electron microscopy. Furthermore, the expression of tcf was correlated with a reduction of the capacity of bacteria to adhere to INT-407 human intestinal epithelial cells in an in vitro assay. In summary, this work demonstrates that the putative adhesion systems found in S. Typhi can indeed be expressed and this expression can be regulated by environmental signals. Furthermore, tcf encodes for a functional fimbria which has never before been observed. Taken together, our results suggest a significant contribution of the putative adhesion systems during normal pathogenesis.

<p>Multiple studies have demonstrated atmospheric cold plasma (ACP) as an effective non-thermal technology for microbial decontamination, surface modification, and functionality alteration in food processing and packaging. ACP constitutes charged particles, such as positive and negative ions, electrons, quanta of electromagnetic radiation, and excited and non-excited molecules, which corresponds to its predominant reactive properties. However, in many of these applications, the interactions between plasma and the components in food matrix are not well-understood. The <b>overall goals</b> of this dissertation were to 1) evaluate the interactions between high voltage atmospheric cold plasma (HVACP) and microbes in liquid and semi-solid food; 2) investigate plasma transfer into semi-solid foods and determine the relationship between microbial inactivation and plasma transfer; 3) explore the interactions between plasma and proteins. </p> <p>The first study explored the microbial (<i>Salmonella</i> <i>enterica</i> serovar Typhimurium, <i>S</i>. <i>enterica</i>) inactivation efficacy of HVACP. The physicochemical interactions between HVACP and biomolecules, including an enzyme (pectin methylesterase, PME), vitamin C and other components in orange juice (OJ) under different conditions was also evaluated. Both direct and indirect HVACP treatment of 25 mL OJ induced greater than a 5 log reduction in <i>S</i>. <i>enterica</i> following 30 s of treatment with air and MA65 gas with no storage. For 50 mL OJ, 120 s of direct HVACP treatment followed by 24 h storage achieved <i>S</i>. <i>enterica</i> reductions of 2.9 log in air and 4.7 log in MA65 gas. An indirect HVACP treatment of 120 s followed by 24 hours storage resulted in a 2.2 log reduction in air and a 3.8 log reduction in MA65. No significant (<i>P </i>< 0.05) Brix or pH change occurred following 120 s HVACP treatment. HVACP direct treatment reduced vitamin C content by 56% in air and PME activity by 74% in air and 82% in MA65. These results demonstrated that HVACP can significantly reduce <i>Salmonella</i> in OJ with minimal quality degradation.</p> <p>The second study in this dissertation examined the penetration process of plasma into semi-solid food and the resulting microbial inactivation efficacy. Agar gels of various densities (0.25, 0.5, 1.0, and 2%) with a pH indicator were inoculated with <i>S</i>. <i>enterica</i> (10⁷>CFU) and exposed directly (between the electrode) or indirectly (adjacent to the plasma field created between the two electrodes) to 90 kV at 60 Hz for up to 1.5 h. A long treatment time (1.5 h) caused sample temperature to increase 5~10 °C. The microbial analysis indicated a greater than 6 log₁₀ (CFU) reduction (both with air and MA65) in the zone with a pH change. Inactivation of bioluminescence cells in the plasma penetrated zone confirmed that the plasma, and its generated reactive species, inactivate microbial as it penetrates into the gel. A two-minute HVACP direct treatment with air at 90 kV induced greater than 5 log₁₀ (CFU)<i> S</i>. <i>enterica </i>reduction in applesauce. <em></em></p> <p>The third study investigated the interactions between HVACP and protein, using bovine serum albumin (BSA) as a model protein. The physicochemical and structural alteration of BSA and its reaction mechanism, when subjected to HVACP, were investigated. After treating 10 mL of BSA solution (50 mg/mL) at 90 kV for 20, 40, or 60 min, we characterized structural alteration and side-group modification. FTIR spectroscopy, Raman spectroscopy, and circular dichroism analysis indicated protein unfolding and decreased secondary structure (25 % loss of α-helix, 12% loss of β-sheet) in HVACP treated BSA. Average particle size in the protein solutions increased from 10 nm to 113 µm, with a broader distribution after 60 min HVACP treatment indicating protein aggregation. SDS-PAGE and mass spectrometer analysis observed a formation of new peptides of 1 to 10 kDa, indicating that the plasma triggered peptide bond cleavage. Chemical analysis and mass spectrometer results confirmed the plasma modifications on the side chains of amino acids. This study reveals that HVACP treatment may effectively introduce structural alteration, protein aggregation, peptide cleavage, and side-group modification to proteins in aqueous conditions, through several physicochemical interactions between plasma reactive species (reactive oxygen species and reactive nitrogen species) and the proteins. This finding can be readily applied to other plasma-protein studies or applications in the food system, such as enzyme inactivation or protein-based film modifications.</p>

T lymphocytes are an essential component of the adaptive immune response and are highly versatile in function. Each T cell has a unique T cell receptor that can recognize an antigenic peptide in the context of the major his to compatibility complex (MHC) encoded molecules, thus offering a high degree of specificity to the immune response. T cells play a central role in the development of an effective host immune response and the quantitative and qualitative regulation of the T cell response is critical. T cells develop in the thymus, an important primary immune organ, where immature thymocytes undergo differentiation and maturation. Through the process of thymic differentiation, immature cluster of differentiation (CD)4-CD8- thymocytes progress to a CD4+CD8+ stage and are subjected to positive and negative selection to give rise to MHC restricted, single positive CD4+ or CD8+ naive T cells that emigrate from the thymus and populate the peripheral lymphocyte pool. Thymic atrophy is well known to occur naturally during the process of aging with thymocyte depletion and reduced thymic output. Along with age associated changes leading to atrophy, the thymus is exquisitely sensitive to starvation and several stresses. In addition, thymic atrophy is a characteristic feature during several viral, bacterial and parasitic infections. Egress of immature thymocytes, loss of thymic populations due to sensitivity to glucocorticoids and cytokine modulation, etc. have been variously proposed to be involved in this process. However there is limited understanding on the numerous mechanisms involved and the crosstalk between these diverse pathways. In this study, a model for thymic atrophy during acute Salmonella enterica serovar Typhimurium (S. typhimurium) infection was developed. S. typhimurium is a Gram negative bacterium that resides and grows in intracellular compartments within host cells. It causes gastroenteritis in humans but leads to typhoid like disease in mice, similar to that caused by S. typhi in humans. Initially, it was established that acute infection of C57BL/6 mice with 108 CFU S. typhimurium, via the oral, i.e. the physiological, route of infection leads to extensive depletion (8-10 fold) of thymocytes in an infection-dependent manner. Infected mice had higher CFU burden in the Peyer’s patches, spleen, liver, and mesenteric lymph node (MLN) as compared to the thymus. The thymic atrophy was dependent upon the infection caused by live S. typhimurium since oral feeding of mice even with higher doses (1010 CFU) of heat-killed bacteria did not lead to thymic atrophy. The susceptible populations in the thymus were identified by staining for expression of CD4 and CD8 on cell surface using specific monoclonal antibodies tagged to fluorophores, e.g. Fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively. The double labelled samples were analyzed by flow cytometry. Interestingly, significant death of CD4+CD8+, the major population of thymocytes, but not single positive thymocytes or peripheral lymphocytes (MLN and spleen cells), was observed at later stages during infection. To gain greater understanding of the processes involved, the mechanisms leading to thymic atrophy were investigated. To this purpose, small molecule inhibitors and mice lacking key molecules important for the immune response were utilized. Also, various assays to assess death of thymocytes, including analysis of death markers such as Annexin V based detection of membrane flipping and caspase activation were performed. I. The extrinsic death pathway involving Fas/FasL interactions is a major death pathway. Therefore, the expression and functional role of the components of the pathway in this model of thymocyte death was investigated. It was observed that thymocytes from infected mice expressed more Fas and Fas ligand (FasL) on their surface than cells from uninfected mice. To address the role of the death receptor, Fas, infection studies were performed with lpr mice that lack functional Fas expression. The depletion of CD4+CD8+ thymocytes in lpr mice was comparable to that in C57BL/6 mice indicating that it was independent of the Fas pathway. However, extensive loss of mitochondrial membrane potential was observed upon analysis with mitochondrial potential specific dyes MitoTracker Red and DiOC6. Most likely, the intrinsic death pathway involving mitochondrial depolarization is involved in this model of thymic atrophy. II. Since thymocytes are known to be sensitive to glucocorticoids both in vitro and in vivo, the involvement of the same in this model of thymic atrophy was assessed. The amounts of cortisol, a glucocorticoid, as detected by ELISA, were elevated during infection. To investigate the functional implication of the increase in cortisol, studies were performed using RU486, a glucocorticoid receptor antagonist. RU486 did not modulate cortisol amounts and treatment of mice with RU486 did not affect CFU burden or survival of mice. However there was a moderate rescue in the number of viable CD4+CD8+ thymocytes, with only a 3-4 fold drop as compared to the 8-10 fold drop in vehicle treated infected mice. III. As glucocorticoids appeared to play a partial role in this model, it was reasonable to assume that other pathways were also involved in the thymic atrophy. The quantitative and qualitative modulation of the cytokine milieu has a profound effect upon the thymus. In fact, inflammatory cytokines, Tnfα and Ifnγ, increased upon infection. In order to study the role of Ifnγ mediated inflammatory responses in this model, infection studies with Ifnγ-/- mice were performed. Ifnγ-/- mice had higher CFU and lower survival; however the drop in thymocyte numbers was 3-4 fold as compared to the 8-10 fold drop in the infected C57BL/6 mice, again indicating a partial involvement of the Ifnγ mediated pathways. In order to study the interactions, if any, between the two pathways mentioned above, corticosteroid signaling was blocked in the Ifnγ-/- mice with RU486. Upon infection, the number of CD4+CD8+ thymocytes was significantly higher in Ifnγ-/- mice treated with RU486 (~1.5 fold drop in viable thymocyte numbers) along with lower caspase 3 activity and mitochondrial damage. Importantly, cortisol amounts in infected Ifnγ-/- mice were comparable to those in infected C57BL/6 mice and the administration of RU486 did not modulate Tnfα and Ifnγ cytokine amounts in sera. Thus, the glucocorticoid and Ifnγ mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S. typhimurium nfection. IV. Although thymic atrophy is known to occur, a detailed characterization of cell surface changes in thymocyte populations has not been performed. To investigate this aspect, thymocytes and MLN cells from uninfected and infected animals were stained for cell surface expression of CD3, CD4, CD5, CD8, CD24, CD25, CD44, CD69, MHC I and MHC II. This analysis was initially performed by studying the changes in expression of these molecules within the total thymocyte and MLN populations. Although there was no change in the expression of CD25 and MHC II in the total thymocyte population upon infection, CD24 expression reduced, whereas, the expression of CD3, CD5, CD44, CD69 and MHC I increased. Notably, changes in the frequency of expression of CD3, CD69 and MHC I were observed before the development of extensive thymic atrophy. The depletion of majority of the CD4+CD8+ thymocytes enriches the mature CD4+ or CD8+ thymocyte population This was corroborated with the observation that, upon in vitro stimulation with PMA and Ionomycin (pharmacological agents used to activate T cells) the residual thymocytes from infected mice produced more IL2 compared to thymocytes from uninfected mice. Subsequently, cells were stained with anti-CD4-FITC, anti-CD8-PE and a third biotinylated antibody, which was detected by a streptavidin-APC conjugate, against one of the remaining six markers. This three colour analysis made it possible to determine the changes in the expression of the third marker in each of the CD4-CD8-, CD4+CD8+, CD4+ and CD8+ populations upon infection. Distinct differences were observed in the phenotypes of uninfected and infected CD4+CD8+ thymocytes and the latter were CD3high, CD5high, CD24low, CD69high and MHC Ihigh indicating that the surviving population had a possibly more mature phenotype. Also, the changes in the phenotypes of the thymocyte populations were dependent upon the extent of thymic atrophy as indicated by time course and CFU studies with C57BL/6 and BALB/c mice respectively. Finally, the roles of glucocorticoids, Ifnγ and Nos2 in modulation of expression of these markers during infection were addressed. Interestingly, the expression of CD3, CD24 and MHC class I significantly correlated with increase in the number of surviving thymocytes upon inhibition of glucocorticoids signaling and in Ifnγ-/- mice. The implications of these changes in the thymocyte surface phenotype during thymic atrophy are discussed. V. Finally, the roles of downstream signalling molecules in S. typhimurium induced thymic atrophy were studied. Although the MAP kinase family members, Erk, Jnk and p38 have been implicated to play a role in the positive and/or negative selection of thymocytes during development, their role in infection induced thymocyte depletion has not been studied. Interestingly, the amounts of Jnk and pJnk, but not p38, increased in thymocytes upon infection. Importantly, pJnk amounts increased predominantly in CD3-/low thymocytes during infection. Furthermore, inhibition of Jnk signalling, using a specific inhibitor SP600125, lead to an increase in survival of CD4+CD8+ thymocytes during infection due to multiple reasons: lowering of cortisol, Tnfα and Ifnγ amounts, and better maintenance of thymic architecture. Thus, inhibition of Jnk mediated signaling protected CD4+CD8+ and CD3-/low thymocytes from death during S. typhimurium infection. Overall, the main conclusions of this study are as follows: First, extensive analysis of the surface phenotype of cells during thymic atrophy throws light on the sensitive and resistant thymocyte populations, thus offering a potential predictive marker profile. Second, glucocorticoids, Ifnγ and, importantly, Jnk mediated signaling play functional roles in the death of immature CD4+CD8+ thymocytes during S. typhimurium infection. The mechanistic details uncovered in this study may be important in designing effective strategies for reducing thymic atrophy during other infections. In fact, enhancement of thymic output may lead to greater numbers and diversity of thymic T cell emigrants in the periphery which is likely to enhance host responses during infections.

Total RNA was extracted from chicken oviduct epithelial cells. Avian Beta-defensin (AvBD)-2, -6, and -12 cDNAs were amplified by reverse transcription-PCR and cloned into pRSET A style='msoareast-language:ZH-CN'>, a protein expression vector. The class=SpellE>hexa-histidine-tagged class=SpellE>AvBD peptides were expressed in Escherichia coli (E. coli) BL21(DE3) class=SpellE>plysS and affinity-purified. The antimicrobial activities of the recombinant AvBDs against E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, Salmonella class=SpellE>enterica style='mso-bookmark:OLE_LINK9'> serovar Typhimurium (S. class=SpellE>typhimurium), and Staphylococcus aureus style='msoareast-language:ZH-CN'> (S. aureus) were determined. style='msoareast-language:ZH-CN;mso-bidiont-style:italic'> At 8, 16 and 32 µg/ml, all three rAvBDs killed and inhibited the growth style='msoareast-language:ZH-CN'> of E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, S. typhimurium, and S. aureus. The killing of rAvBD-2, -6, and -12 against stationary phase E. coli and S. class=SpellE>aureus was pH dependent in the range investigated. style='msoareast-language:ZH-CN'> In addition, the killing-curves showed that rAvBDs exerted their antimicrobial function within 30 minutes of treatment, suggesting the fast killing mechanisms of rAvBDs.

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Le genre bactérien Salmonella regroupe plus de 2500 sérovars, mais peu sont responsables de pathologies humaines. Salmonella enterica sérovar Typhi (S. Typhi) est reconnu pour son importance médicale à travers le globe. S. Typhi cause la fièvre typhoïde chez l’Homme, une maladie infectieuse létale caractérisée par la dissémination systémique de la bactérie vers des organes du système réticulo-endothélial. La fièvre typhoïde représente un fardeau pour la santé mondiale, notamment auprès des pays en développement où les conditions sanitaires sont désuètes. La situation se complique davantage par l’apparition de souches résistantes aux antibiotiques. De plus, les deux vaccins licenciés sont d’efficacité modérée, présentent certaines contraintes techniques et ne sont pas appropriés pour les jeunes enfants et nourrissons. La phase systémique de l’infection par Salmonella repose sur sa survie dans les macrophages du système immunitaire. Dans ce compartiment intracellulaire, la bactérie module les défenses antimicrobiennes grâce à de multiples facteurs de virulence encodés dans son génome. Les mécanismes moléculaires sollicités sont complexes et finement régulés. Malgré les progrès scientifiques réalisés précédemment, plusieurs incompréhensions persistent au sujet de l’adaptation de ce pathogène dans les macrophages de l’hôte. Pour mieux concevoir les déterminants génétiques de S. Typhi impliqués dans l’interaction avec ces cellules, une stratégie de sélection négative a été appliquée afin de vérifier systématiquement l’effet direct des gènes pendant l’infection. En premier temps, une librairie de mutants par transposon chez S. Typhi a été créée pour l’infection de macrophages humains en culture. Après 24 heures d’infection, la présence des mutants fut évaluée simultanément par analyse sur des biopuces de Salmonella. Au total, 130 gènes ont été sélectionnés pour leur contribution potentielle auprès des macrophages infectés. Ces gènes comptaient des composantes d’enveloppe bactérienne, des éléments fimbriaires, des portions du flagelle, des régulateurs, des facteurs de pathogenèse et plusieurs protéines sans fonction connue. En deuxième temps, cette collection de gènes a dirigé la création de 28 mutants de délétion définie chez S. Typhi. Les capacités d’entrée et de réplication intracellulaire de ces mutants au sein des macrophages humains ont été caractérisées. D’abord, les macrophages ont été co-infectés avec les mutants en présence de la souche sauvage, pour vérifier la compétitivité de chacun d’eux envers cette dernière. Ensuite, les mutants ont été inoculés individuellement chez les macrophages et leur infectivité fut mesurée comparativement à celle de la souche sauvage. Sommairement, 26 mutants ont présenté des défauts lorsqu’en compétition, tandis que 14 mutants se sont montrés défectueux lorsque testés seuls. Par ailleurs, 12 mutants ont exposé une déficience lors de l’infection mixte et individuelle, incluant les mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346 et SPI-4. Notamment, 35 nouveaux phénotypes défectueux d’entrée ou de survie intracellulaire chez Salmonella ont été révélés par cette étude. Les données générées ici offrent plusieurs nouvelles pistes pour élucider comment S. Typhi manipule sa niche intracellulaire, menant à l’infection systémique. Les gènes décrits représentent des cibles potentielles pour atténuer la bactérie chez l’humain et pourraient contribuer au développement de meilleures souches vaccinales pour immuniser contre la fièvre typhoïde. / The bacterial genus Salmonella holds over 2500 serovars, but few are responsible for human pathologies. Salmonella enterica serovar Typhi (S. Typhi) is recognized across the globe for its medical importance. S. Typhi causes typhoid fever in humans, a lethal infectious disease characterized by systemic dissemination of the bacteria to organs of the reticulo-endothelial system. Typhoid fever represents a burden for public health, notably in developing countries where sanitary conditions are obsolete. The situation is further complicated by the appearance of strains resistant to antibiotics. Moreover, both of the licensed vaccines are of moderate efficiency, present certain technical constraints and are not appropriate for young children and newborns. The systemic phase of infection by Salmonella relies on its survival within macrophages of the immune system. In this intracellular compartment, the bacterium modulates antimicrobial defenses thanks to multiple virulence factors encoded within its genome. Molecular mechanisms taking place are complex and finely regulated. Despite scientific advances made previously, many misunderstandings persist concerning the adaptation of this pathogen within host macrophages. To better conceive the genetic determinants of S. Typhi involved in interaction with these cells, a negative selection strategy was applied to systematically verify the direct effect of genes during infection. Firstly, a library of transposon insertion mutants in S. Typhi was created for infection of cultured human macrophages. After 24 hours of infection, the presence of mutants was evaluated simultaneously by analysis on Salmonella microarrays. In total, 130 genes were selected for their potential contribution within infected macrophages. These genes included bacterial envelope components, fimbrial elements, portions of the flagellum, regulators, pathogenesis factors, and many proteins of unknown function. Secondly, this collection of genes led to the creation of 28 defined deletion mutants in S. Typhi. The ability of entry and intracellular replication of these mutants within human macrophages were characterized. To start, macrophages were coinfected with mutants in the presence of the wild-type strain, in order to verify the competitiveness of each of them against the latter. Then, mutants were inoculated individually into macrophages and their infectiveness was measured in comparison with the wild-type strain. In summary, 26 mutants presented defects when in competition, whereas 14 mutants were shown defective when tested alone. Furthermore, 12 mutants exposed a deficiency during mixed and individual infection experiments, including mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346, and SPI-4. In particular, 35 new defective phenotypes of Salmonella entry or intracellular survival were revealed in this study. Data generated here provides significant novel insight for elucidating how S. Typhi manipulates its intracellular niche, leading to systemic infection. Genes described represent potential targets for attenuating the bacteria in the human host and could contribute to the development of better vaccine strains to immunize against typhoid fever.

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