L’invasion de la muqueuse du côlon humain par les entérobactéries à Gram négatif du genre Shigella aboutit à une rectocolite aigue appelée dysenterie bacillaire qui reste un problème de santé publique majeur. Shigella possède un Système de Sécrétion de Type Trois (SST3) codé par un plasmide de virulence qui permet la translocation d’effecteurs bactériens dans le cytoplasme des cellules eucaryotes pour manipuler leurs fonctions. Ces effecteurs détournent les cellules épithéliales pour constituer une niche de réplication intracellulaire et interagissent avec les cellules immunes pour affecter l’initiation de la réponse immune adaptative. En conséquence, plusieurs épisodes infectieux sont nécessaires afin d’établir une protection immunitaire humorale qui est toutefois de courte durée. Ces travaux de thèse avaient pour but (i) d’approfondir les connaissances sur l’interaction de Shigella avec l’hôte en se concentrant sur les mécanismes dépendant du SST3 interférant avec les lymphocytes et (ii) de déterminer si des effecteurs du SST3 non encore identifiés sont codés par le plasmide de virulence. Nos résultats démontrent que la translocation d’effecteur du SST3 peut être découplée de l’invasion cellulaire, conduisant à des cellules « injectées-seulement ». Nous rapportons que Shigella induit l’apoptose des lymphocytes B par un mécanisme dépendant de la protéine située à l’extrémité du SST3 mais indépendant de la translocation d’effecteurs. Ces résultats établissent un nouveau paradigme concernant la pathogénicité de Shigella au-delà de l’invasion cellulaire, basé sur des mécanismes de type « touché-coulé » qui sont au cœur des interactions entre ce pathogène et les cellules immunes. Par ailleurs, nous décrivons la capacité de Shigella jusque-là inconnue d’interférer avec la sécrétion d’anticorps par les lymphocytes B, ce qui pourrait contribuer à moduler la réponse spécifique humorale de l’hôte. Enfin, nous avons identifié cinq nouveaux effecteurs potentiels de Shigella codés par le plasmide de virulence et injectés par le SST3 dans les cellules eucaryotes. Ces travaux de thèse apportent donc de nouveaux éléments concernant la pathogénicité de Shigella par la découverte de nouveaux mécanismes ciblant les cellules immunes et l’identification de nouvelles protéines bactériennes injectées dans le cytoplasme des cellules de l’hôte. / Invasion of the human colonic mucosa by the Gram-negative enterobacteria Shigella spp. results in an acute recto-colitis named bacillary dysentery that still remains a major public health concern. Shigella expresses a Type Three Secretion System (T3SS) encoded on a virulence plasmid and mediating translocation of bacterial effectors into eukaryotic cell cytoplasm to manipulate their functions. These effectors hijack epithelial cells to create a bacterial intracellular replicative niche and also interact with immune cells to affect the priming of the adaptive immune response. As a result, several episodes of infection are required to mount a protective humoral immunity that is nevertheless of short-duration. This thesis work aimed at (i) further documenting Shigella cross-talks with its host, with a particular focus on T3SS-mediated mechanisms towards lymphocytes and (ii) investigating if the Shigella virulence plasmid encodes for yet unidentified T3SS-effectors. We report that translocation of Shigella T3SS-effectors into lymphocytes can be uncoupled from cellular invasion, resulting in “injected-only” cells. We demonstrate that Shigella mediates B lymphocyte apoptosis through a mechanism depending on the secretion apparatus needle tip protein but independent from effectors translocation. These findings set up a new paradigm for Shigella pathogenicity beyond cellular invasion, with “kiss-and-run” mechanisms proposed to be at the core of the interactions between this pathogen and immune cells. In addition, we describe a so far not known capacity of Shigella to interfere with B lymphocyte antibody secretion that could contribute to divert Shigella- specific humoral immunity. We also identify five new putative Shigella effectors encoded by the virulence plasmid and translocated by the T3SS into eukaryotic cells. Thus, this thesis work brings new insights into Shigella pathogenicity by unraveling novel mechanisms towards host immune cells and identifying new bacterial proteins that might constitute additional molecular weapons for this pathogen.

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacteria responsible for significant morbidity and mortality in young children. EPEC elaborates a type III secretion system (T3SS), which translocates bacterial effector proteins into the host intestinal epithelial cell. To this date, 23 effector proteins are known to be secreted by EPEC. Over the past two decades, traditional studies uncovered the functions of some of these effector proteins. While there was an initial rise in the EPEC effector function discoveries, we now observe a plateau in the identification of host-EPEC interactions. Thus, the aim of my dissertation is to define novel virulence strategies in EPEC pathogenesis, and to demonstrate how traditional reductionist and global systems biology approaches can be utilized in uncovering functions of individual effectors, as well as the complex interplay of effectors in modulating host functions. Specifically, we defined the novel cytoprotective function of a T3SS effector EspZ. We further illustrated the complex interplay of EPEC effectors by defining how EPEC utilizes EspZ and EspF to dynamically regulate the prosurvival epidermal growth factor receptor signaling pathway. Finally, by integrating comparative proteomics and traditional reductionist approaches, we identified a novel function for EspH, and defined the mechanism by which EspH perturbs epithelial cell structure and function.

No description available.

theresa.j.ochoa@uth.tmc.edu / Article / BACKGROUND: Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. METHODS: EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. RESULTS: The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77-1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10-87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). CONCLUSIONS: EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Acute diarrhea induced by Escherichia coli is an important illness in humans, especially in children under age of two in developing countries. Citrobacter rodentium is used as murine model for E. coli infection in humans because it causes ultrastructural changes in murine colonic epithelium comparable to lesions produced by enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Adult mice of many strains develop self-limiting epithelial hyperplasia when infected, whereas adult C3H and FVB mice are highly susceptible to infection and demonstrate mortality rates between 60 and 100% two weeks after infection. These susceptible strains of mice also have higher bacterial translocation to mesenteric lymph nodes. In mice, the cause of death could be hypovolemia due to dehydration that may occur due to an increase in paracellular permeability as well as dysregulation of apical and basolateral ion transporting proteins. C. rodentium virulence factors resemble those of E. coli and are believed to primarily alter tight junctions of colonic epithelial cells. Effectors delivered via the type III secretory system have been associated with actin condensation and pedestal formation. The exact mechanisms of C. rodentium infection, as well as changes that occur in vitro as well as in the intestine of various strains of mice are not completely understood. This study introduced a new in vitro Ptk6 cell line for C. rodentium infection, which can also serve as a model for EPEC in humans. Effect of C. rodentium on colonic epithelial cells of susceptible and resistant mice was determined in in vivo study. C. rodentium attached to Ptk6 colonic epithelial cells, inducing attaching and effacing (A/E) lesions and loss of monolayer integrity, which charachterizes this cell line as a relevant in vitro model of C. rodentium and EPEC infections. Murine studies revealed that C. rodentium induced more severe disease and 100% mortality in juvenile C3H mice whereas Swiss Webster (SW) mice expressed only moderate morbidity. The colonic lesions and changes in barrier function of colonic epithelium were more prominent in C3H mice. This study determined potential targets in the murine colon that play role the establishment and the outcome of the infection, indicating multifactorial nature of C. rodentium-induced diarrhea. This study identified host factors involved in the initiation of C. rodentium-associated diarrhea and the outcome of infection, which can be useful in developing of novel strategies for preventing and treatment of infectious colitis.

Neonates are generally thought to be more likely to suffer from gastrointestinal disease, owing in part to diminished immune cell function. To gain insight into the development of mucosal immune responses during early life, we developed a model of orogastric infection with the Gram-negative bacterium Yersinia enterocolitica using murine neonates. Remarkably, neonatal mice of either the BALB/c or C57BL/6 mouse strains showed markedly enhanced survival after infection compared to adult mice. Both innate and adaptive immune components appear to contribute to this phenomenon. First, the increased resistance of neonates coincided with containment of the bacteria in the intestinal tissue with low dissemination into the spleen and liver. In contrast, the bacteria readily disseminated to the peripheral tissues in adult mice. Flow cytometric and histological studies revealed increased levels of neutrophils and macrophages in the neonatal mesenteric lymph nodes (MLN) compared to adult mice. Similar results were obtained using two different high virulence Y. enterocolitica strains. The rapid mobilization of innate cells sequestered the bacteria to the intestinal tissue, since in vivo neutrophil depletion led to efficient dissemination of Y. enterocolitica to the spleen and liver of neonates. Together, these results support the hypothesis that the neonatal intestinal immune system is competent to mount a strong antibacterial response by rapidly mobilizing innate phagocytes and thereby confining the bacterial infection to the gut, resulting in a high level of resistance. Second, we have also demonstrated that the adaptive immune system was mobilized during primary and secondary infection with this pathogen and that some of these factors may contribute to the enhanced resistance of neonatal mice to infection. Primary infection in neonates led to increased levels of antigen presenting cells, B and T cells with an activated phenotype in the MLN. MLN CD4+ Th cells from infected neonates were found to produce greater levels of IFN-gamma and IL-17A, compared to CD4+ Th cells from adult mice. These Th responses are likely to be functionally significant because neonatal mice deficient in CD4+ T cells were found to be more susceptible than adult mice to primary infection. CD4+ T cells adoptively transferred into CD4 deficient mice rescued the majority of mice from lethal infection and led to the production of IFN-gamma and IL-17A by MLN cells. In addition, primary T cell-dependent IgG1 and IgG2a serum antibodies specific for the Yersinia immunogen LcrV were increased compared to adult mice, and the absence of B cells partially increased the susceptibility of neonatal mice to primary infection. During secondary infection, however, neonatal and adult mice mounted quantitatively and qualitatively similar Yersinia-specific memory antibody responses, demonstrating that infection with Y. enterocolitica promotes mature B cell responses in neonatal mice. Finally, primed neonatal and adult mice were protected from colonization of the Peyer's patches, weight loss and mortality after a lethal infection in adulthood, demonstrating the development of long-lived protective memory responses at the intestinal interface. Together, these results indicate that both B and T cell responses, in particular Th1 and Th17 associated immunity, are important for the development of long lasting immunity to this pathogen in early life. Third, infection of neonatal mice with a Y. enterocolitica strain deleted of the anti-inflammatory protein YopP led to massive infiltration and/or accumulation of innate phagocytes in the intestine and MLN. This effect was not detectable in infected adult mice. Thus, we have identified a novel negative regulator of intestinal inflammation which might be valuable in preventing or ameliorating inflammatory conditions. This model system has revealed the unprecedented potential of neonatal mice to develop protective inflammatory innate and adaptive immunity at mucosal surfaces. The combined results presented here demonstrate that neonatal mice may be well equipped to mount robust innate and adaptive intestinal inflammatory responses that are highly protective toward Y. enterocolitica. These findings have implications for understanding how pediatric intestinal adaptive immune responses develop in response to naturally occurring gastroenteric pathogens and offer a new biological platform for development of vaccines aimed at improving mucosal and systemic immunity in early life.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Escherichia coli enteropatogÃnica (EPEC) à um importante patÃgeno associado Ãs doenÃas diarreicas. InfecÃÃes intestinais ocasionam comprometimento da barreira intestinal e um dos primeiros mecanismos de resposta à recuperaÃÃo à a migraÃÃo das cÃlulas intestinais. As principais proteÃnas que regulam esse processo sÃo as pequenas GTPases Rho, Rac1, RhoA e Cdc42A. A alanil-glutamina (Ala-Gln) estimula este processo migratÃrio, entretanto os mecanismos envolvidos nesta resposta ainda sÃo desconhecidos. Desse modo, investigou-se o efeito de uma cepa selvagem e padrÃo (E2348/69) de EPEC, e de uma cepa comensal E. coli HS na migraÃÃo celular intestinal, bem como a regulaÃÃo da transcriÃÃo e expressÃo gÃnica das GTPases Rho e o papel da suplementaÃÃo com Ala-Gln no processo de migraÃÃo na presenÃa ou ausÃncia da infecÃÃo. A infecÃÃo pelas cepas de EPEC e pela cepa comensal reduziram significativamente a migraÃÃo celular intestinal. Entretanto, houve uma maior reduÃÃo desse efeito nas cÃlulas infectadas pelas cepas de EPEC quando comparado Ãquelas infectadas pela cepa comensal de E. coli HS. Observou-se um alto percentual de cÃlulas necrÃticas, cerca de 30%, induzido pela cepa padrÃo de EPEC apenas nos tempo de 12 e 24 horas apÃs infecÃÃo. A adiÃÃo da Ala-Gln em cÃlulas nÃo infectadas estimulou significativamente e de modo dose dependente a migraÃÃo apÃs 24 horas. PorÃm, quando esse nutriente foi adicionado durante 12 e 24 horas na presenÃa da infecÃÃo, nÃo houve uma reversÃo do dano. Em relaÃÃo à expressÃo gÃnica das GTPases Rho, observou-se um aumento da transcriÃÃo de rac1 nas cÃlulas que haviam sido infectadas pelas cepas de EPEC e E. coli HS, bem como um aumento da transcriÃÃo de rhoA nas cÃlulas infectadas pela cepa padrÃo de EPEC apÃs 2 horas da infecÃÃo. Todavia, na anÃlise das proteÃnas por imunofluorescÃncia, RhoA e Cdc42 mostraram-se aumentadas nas cÃlulas infectadas pela EPEC padrÃo quando comparado ao controle. Enquanto que as cÃlulas infectadas com a cepa selvagem de EPEC observou-se um aumento de Rac1 e reduÃÃo de RhoA. Esses dados mostraram que a migraÃÃo das cÃlulas intestinais à reduzida principalmente pelas cepas patogÃnicas de EPEC, ao regular a transcriÃÃo e expressÃo gÃnicas das proteÃnas GTPases Rho. A suplementaÃÃo com Ala-Gln em cÃlulas intestinais promoveu a migraÃÃo celular apenas na ausÃncia da infecÃÃo. / Enteropathogenic Escherichia coli (EPEC) is an important pathogen associated with diarrheal diseases. Intestinal infections cause impairment of the intestinal barrier and one of the earliest response mechanisms to recover is migration of the intestinal cells to cover the injured area. The key proteins that regulate cell migration are small Rho GTPases, Rac1, Cdc42 and RhoA. The alanyl-glutamine (Ala-Gln) increases this migration process, however the mechanisms involved in this response are still unknown. Thus, we investigated the effect of a wild type strain and standard (E2348/69) of EPEC strain and a commensal E. coli HS on intestinal cell migration, as well as transcriptional regulation and gene expression of Rho GTPases and the role of supplemental Ala-Gln in the migration process in the presence or absence of infection. Infection by EPEC strains and commensal E. coli HS significantly reduced intestinal cell migration. However, this effect was more pronounced in cells infected by the strains of EPEC compared to those infected by the commensal strain of E. coli HS. We observed a high percentage of necrotic cells, about 30%, induced only by EPEC strain pattern 12 and 24 hours after infection. The addition of Ala-Gln in uninfected cells significantly stimulated in a dose dependent migration after 24 hours. However, when this nutrient was added over 12 and 24 hours in the presence of infection, there was no reversion of the damage. Regarding the gene expression of Rho GTPases, we observed an increase in transcription of rac1 in cells that had been infected by the strains of EPEC and E. coli HS as well as an increase in rhoA transcription in cells infected with EPEC strain pattern at 2 hours after infection. However, the analysis of proteins by immunofluorescence, RhoA and Cdc42 shown to be elevated in cells infected with EPEC pattern when compared to the control. Whereas cells infected with wild EPEC strain was observed an increase of Rac1 and reduction of RhoA. These data showed that cell migration is reduced mainly by the intestinal pathogenic strains of EPEC, in regulating gene transcription and expression of the protein Rho GTPases. Supplementation with Ala-Gln in intestinal cells only promoted cell migration in the absence of infection.

Les bactéries entéropathogènes du genre Salmonella et Shigella sont transmises par les aliments ou l’eau et sont responsables de nombreuses infections entériques chez les animaux et les humains. Ces maladies infectieuses restent une cause importante de morbidité et de mortalité dans les pays en voie de développement. L’existence de multiples sérotypes de Salmonella et de Shigella ainsi que l’émergence de souches résistantes aux antibiotiques, nécessite le développement de vaccins efficaces et large spectre. Ces bactéries utilisent un système d’injection de leurs protéines effectrices, appelé injectisome ou encore Système de Sécrétion de Type III (SST3), nécessaire à leur pathogénicité. Alors que les protéines effectrices injectées au moyen de cet injectisome sont variées et dépendent essentiellement du type cellulaire cible et donc de la spécificité du pathogène, certaines des protéines structurales composant l’injectisome sont relativement bien conservées parmi les différentes bactéries pathogènes, notamment les protéines de l’aiguille : PrgI et MxiH, et celles de la coiffe de l’aiguille : SipD et IpaD, respectivement de Salmonella et Shigella. Ces protéines, fortement impliquées dans la virulence des bactéries, semblent donc être des cibles de choix pour lutter contre des infections opportunistes impliquant ces bactéries pathogènes.Le premier objectif de cette thèse était d’évaluer l’immunogénicité et l’effet protecteur des protéines structurales de l’injectisome citées précédemment contre les infections à Salmonella et Shigella. Les protéines recombinantes préparées et produites au laboratoire ont été utilisées de façon séparée ou en combinaison pour immuniser des souris par différentes routes. Ensuite, les réponses immunitaires des souris ainsi immunisées ont été analysées par des tests immunométriques. Enfin, le potentiel immunogène et vaccinant de ces protéines structurales a été évalué en infectant les souris immunisées avec 100 DL50 de Salmonella par voie orale ou de Shigella par voie intranasale. Le meilleur résultat a été obtenu en utilisant la voie intra-gastrique pour les immunisations avec environ 70% de protection. Cette stratégie a permis également d’évaluer la pertinence de cette approche vaccinale dans un modèle murin de protection croisée (entre 25 et 60%). Le deuxième objectif de cette thèse était d’évaluer le pouvoir protecteur d’anticorps monoclonaux murins reconnaissant les régions conservées des protéines SipD et IpaD. Les anticorps obtenus ont été caractérisés et leur pouvoir neutralisant a été évalué in vivo dans un modèle murin d’infection avec Salmonella ou Shigella (jusqu’à 60% de protection).L’ensemble de ces travaux montre que l’utilisation de certaines protéines structurales conservées de l’injectisome de bactéries entéropathogènes présente un intérêt vaccinal et immunothérapeutique pour aider au traitement de certaines salmonelloses et shigelloses. / Salmonella and Shigella species are food and water borne pathogens that are responsible for enteric infections in both humans and animals. These infectious diseases are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of antibio- resistant strains, require the development of protective and broad-spectrum vaccines. All these bacteria utilize a system for injection of their effectors, called injectisome or Type III Secretion System (T3SS), necessary for their pathogenicity. While effector proteins are varied and depend essentially on the cellular target and thus on the specificity of the pathogen, the structural proteins that form the injectisome are common to all virulent Salmonella and Shigella spp., particularly the needle proteins PrgI and MxiH and the needle-tip proteins SipD and IpaD of Salmonella and Shigella respectively. These proteins, strongly involved in the virulence of the bacteria, appear to be ideal candidate antigens for a subunit-based, broad spectrum vaccine.The first aim of my PhD was to evaluate the immunogenicity and protective efficacy of structural proteins of the above-mentioned injectisome against Salmonella and Shigella infections. The recombinant proteins were prepared and produced in the laboratory and were used alone or in combination to immunize mice using different routes. The immune responses of immunized mice were then analyzed by immunometric assays. Finally, the protective efficacy was evaluated in a mouse model of intestinal (Salmonella) or pulmonary (Shigella) challenge. The best result was obtained by orogastric immunization with 70% of protection. This strategy also allowed to estimate the relevance of this approach in a mouse model of crossed protection (from 25 to 60%). The second objective of my PhD was to evaluate the protective efficacy of murine monoclonal antibodies recognizing conserved regions of SipD and IpaD proteins. The obtained antibodies were characterized and their therapeutic effect was evaluated in vivo with a Salmonella and Shigella infection murine model (up to 60% of protection).To conclude, this work showed that some conserved structural proteins composing the injectisome of enteropathogenic bacteria is of interest for treatment of enteric diseases caused by Salmonella and Shigella.

Yes / Enteropathogenic Escherichia coli (EPEC) is usually associated with outbreaks and sporadic cases of severe infantile diarrhoea in the developing world, and less commonly with sporadic cases in developed countries. Very little evidence indicates that EPEC is a food-borne pathogen for adults. In a previous study, two groups of adult travellers became ill, and eae+ E. coli of serogroup O111 was isolated from affected individuals and epidemiologically linked to food consumption. Here the strain responsible was further investigated and characterized as an unusual atypical EPEC. PCR analysis of the designated type isolate showed the presence of the rorf1 and espB genes of the LEE pathogenicity island, which was inserted at the chromosomal selC locus. The isolate was negative for the enteroaggregative E. coli EAST-1 toxin present in other strains of EPEC associated with food-borne outbreaks. The strain adhered sparsely to HEp-2 cell monolayers in a diffuse manner, but fluorescent actin staining demonstrated that it was capable of inducing polymerization of actin at the sites of bacterial attachment. Strain P2583 is the first EAST-negative EPEC to be confirmed as a cause of outbreaks of infection in adults following the consumption of contaminated food or water.

No description available.