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Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host–pathogen interactions.

Enterohemorrhagic Escherichia coli (EHEC) are a major public health concern. Indeed, these pathogens are responsible for thousands of food-borne illness cases worldwide every year and can lead to serious complications, including kidney damages in young children and brain damages in the elderly. Currently, the main issue is the limited number of available therapeutic treatments since antibiotic therapy can promote the development of infection-related complications. Therefore, it appears essential and topical to identify bacterial factors associated with EHEC virulence and to understand the interactions occurring between the pathogen and the host, in order to develop new anti-infective strategies. The first objective of this thesis was to identify new bacterial factors potentially involved in the infectious process. Application of the RIVET technology to the reference strain O157:H7 EDL933 revealed 31 genes specifically induced during mouse infection. Characterization of these genes showed that some of them encode niche factors potentially involved in the adaptation of EHEC to the intestinal environment, therefore contributing to virulence. The second aim of this thesis was to characterize in vivo the response of EHEC to nitric oxide (NO), a mediator of the host’s immune response, and thus assess the protective role of NO against EHEC infection in a mouse model. By using a NO-sensing reporter EHEC strain, we demonstrated that NO is produced by the host at the early stages of infection and this NO limits adhesion of the pathogen to the colonic mucosa. On the other hand, we also showed that NO is detrimental to the host since it promotes the production of Shigatoxins (Stx), which is the major EHEC virulence factor, and leads to the development of renal dysfunction. Finally, we showed that the NO reductase NorVW is important for the virulence of some, but not all, EHEC strains. Inactivation of the norVW operon in strain O157:H7 620 reduces the ability of the pathogen to efficiently colonize the digestive tract and to produce Stx. However, this observation is strain-specific and this suggests that EHEC response to nitrosative stress during infection is complex and probably multifactorial. This work contributes to a better understanding of the EHEC infectious process, an essential step for the development of future anti-infective strategies., Les Escherichia coli entérohémorragiques (EHEC) représentent un enjeu majeur en santé publique. En effet, ces pathogènes sont responsables chaque année de milliers de cas de toxi-infections alimentaires à travers le monde et peuvent engendrer des complications graves, notamment des atteintes rénales chez les jeunes enfants et cérébrales chez les personnes âgées. Actuellement, le principal problème réside dans le fait que les traitements thérapeutiques disponibles sont limités puisque l’antibiothérapie peut favoriser le développement des complications liées à l’infection. Il est donc primordial et d’actualité de mettre en évidence les facteurs bactériens associés à la virulence des EHEC et de comprendre les interactions entre le pathogène et l’hôte afin de développer des stratégies thérapeutiques visant à éliminer le pathogène et limiter l’apparition des symptômes graves. Ainsi, le premier objectif de cette thèse était d’identifier de nouveaux facteurs bactériens potentiellement impliqués dans le processus infectieux. L’utilisation de la technologie RIVET sur la souche de référence O157:H7 EDL933 en modèle murin, a permis de mettre en évidence 31 gènes dont l’expression est spécifiquement induite lors de l’infection. La caractérisation de ces gènes a démontré que certains codent des facteurs de niche qui pourraient accroître le potentiel des souches d’EHEC à s’adapter à l’environnement intestinal et ainsi participer à la virulence du pathogène. Le second volet de cette thèse avait pour but de caractériser in vivo la réponse des EHEC au monoxyde d’azote (NO), un médiateur de la réponse immunitaire de l’hôte, et ainsi d’évaluer le potentiel rôle protecteur du NO lors d’une infection en modèle murin. En utilisant une souche d’EHEC rapportant la présence de NO, nous avons démontré que le NO est produit dès les premiers stades de l’infection et que celui-ci limite l’adhésion du pathogène à la muqueuse colique. En revanche, nous avons également mis en évidence un effet néfaste du NO pour l’hôte puisqu’il favorise la production des Shigatoxines (Stx), le facteur de virulence majeur des EHEC, conduisant au développement d’un dysfonctionnent rénal. Enfin, nous avons montré l’importance de la NO réductase NorVW dans la virulence de certaines souches d’EHEC. En effet, l’inactivation de l’opéron norVW chez la souche O157:H7 620 réduit la capacité du pathogène à coloniser efficacement le tractus digestif et à produire Stx. Cette observation est toutefois souche dépendante et suggère que la réponse des EHEC au stress nitrosant lors d’une infection est complexe et probablement multifactoriel. L’ensemble de ces travaux contribue à une meilleure compréhension du processus infectieux des EHEC, une étape indispensable au développement de futures stratégies anti-infectieuses.

National audience; Epidemiologic studies reveal that only few virulence factors are frequently associated with Shiga toxin-producing Escherichia coli (STEC) strains that cause severe disease in humans. Among them, Shiga toxins (Stx) is required for the development of the most severe symptoms in STEC infected patients but is not strictly sufficient to make an E. coli strain pathogenic to human. Indeed, many STEC strains isolated from rearing animals have never been implicated in human disease. It therefore appears crucial to increase our knowledge on STEC pathogenesis at the molecular level. We developed a RIVET (Recombination-based in vivo expression technology) strategy in order to identify STEC genes specifically induced in vivo during mouse infection. Construction and screening of a RIVET promoter library from the typical STEC O157:H7 strain EDL933 resulted in the identification of 31 in vivo induced (ivi) genes. Most but not all, have an attributed function and are involved in either metabolism or stress response pathways, indicating that STEC has to adapt to the intestinal ecosystem. Additionally, some identified ivi genes belong to the dispensable genome of E. coli and analysis of their distribution among E. coli strains revealed a strong prevalence for some of them specifically in STEC strains. We also observed correlations between ivi genes and virulence genes, serotypes and/or seropathotypes. Finally, we assessed their contribution to STEC pathogenesis by infecting mice with mutants inactivated for selected ivi genes. This work may thus help to improve STEC molecular risk assessment schemes by giving new insights into molecular aspects of STEC pathogenesis.

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Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host–pathogen interactions.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host–pathogen interactions.