Enterohaemorrhagic Escherichia coli (EHEC) are a recently emerged group of pathogens that can cause fatal infections in the young and elderly. EHEC utilize a virulence factor delivery organelle called a ’Type 3 secretion system’ that results in the formation of characteristic ‘pedestal structures’ on epithelial cells allowing colonization in the human or ruminant gastrointestinal tract. To achieve this, effector proteins have to be injected into host cells. The SepL-SepD complex has been shown to be key for controlling T3-related protein secretion in EHEC. Lack of either protein results in effector hypersecretion and strongly impaired secretion of EspADB translocon proteins. Therefore, the expression and function of SepL was the focus of my PhD research. The expression of SepL was shown to be heterogeneous and co-expressed with EspA filaments in EHEC O157 strains. My work revealed two transcriptional regulators (Ler and SepD) and two putative posttranscriptional regulators (Hfq and CsrA) of SepL expression. Further experiments mapped a key mRNA region required for heterogeneous expression of SepL. This sequence forms a predicted hairpin structure around the Shine-Dalgarno (SD) site of sepL. A model has been formed based on my data in which Hfq and CsrABCD bind to the mRNA potentially competing to control translation. Functionally, the C-terminus of SepL was found to be expendable for 1) SepD binding; 2) SepL membrane localization and 3) translocon export, however it was required for 1) limiting effector secretion via (2) a Tir interaction which might be disassociated by (3) an EscD interaction once host cell signals are sensed. Previously, the concept of two different types of T3 secretion signal were demonstrated in Yersinia spp, I tested this hypothesis in EHEC using both wild type and SepL/SepD deficient EHEC strains. SepL/SepD is required for the N-terminal signal pathway but not a chaperone binding domain signal pathway. A 12aa NleA which only contained an N-terminal signal was shown to bind to SepD and so did the multi-functional T3 chaperone ― CesT. Finally, Far-Western assays demonstrated that SepL only interacted with Tir while SepD could bind other effector proteins indicating that SepL/SepD may act as a targeting hub for effector protein secretion.

Escherichia coli enterohemorrágicas (EHEC) que possuem a Ilha de Patogenicidade LEE são importantes patógenos humanos. A habilidade de adesão, invasão e perfil genético têm sido estudados, já que sorotipos que não possuem LEE tem sido isolados de pacientes com doença severa e intracelularmente. Das nove amostras relacionadas com doença, quatro (44.5%) apresentaram stx2+. Todas foram positivas para o gene lpfA e iha, e negativas para toxB. Três (33,3%) apresentaram saa e cinco hly. A maioria apresentou padrão de adesão difusa e invasão negativa em células HEp-2. Em CaCO-2 apresentaram aderência com variados graus de intensidade, e a maioria das amostras testadas apresentou invasão maior que 3,3%. Frente ao inibidor de polimerização de actina citocalasina D, houve significativa redução nos níveis de invasão, sugerindo que estas amostras utilizam um mecanismo da célula hospedeira para internalização, e que provavelmente, fatores de virulência como adesinas, favorecem a adesão das mesmas, compensando a ausência do LEE, e facilitando a instalação da infecção. / Enterohaemorrhagic Escherichia coli (EHEC) that possess the pathogenic island LEE are important human pathogens. The adhesion ability, invasion and genetic profile have been studied, since serotypes that do not possess LEE have been isolated from humans with severe disease and found intracellular. Nine strains related with SHU, four (44,5%) were stx2+. All strains were positive for IpfA and iha genes and negative for toxB. Three (33,3%) showed saa and five hly. The most strains showed a diffusely adhesion pattern and negative invasion in HEp-2 cells. It presented various degrees of adhesion, and the most tested strains showed invasion high than 3,3% in CaCO-2. That was a significant reduction of invasion in the presence of actin polymerization inhibitor Citochalasin D, suggesting that these strains use a host cell mechanism to invade, and probably virulence factors, like adhesins, favors this adhesion and compensate LEE absence, promoting the installation of infection.

Les Escherichia coli entérohémorragiques (EHEC) O157:H7 sont des agents pathogènes alimentaires entrainant des colites hémorragiques et des syndromes hémolytiques et urémiques (SHU). Depuis le ruminant, le réservoir naturel, les EHEC peuvent contaminer certaines denrées alimentaires et in fine infecter l’Homme. La présence de composants de la matrice extracellulaire (ECM) le long de la chaine alimentaire pourrait participer à leur capacité de colonisation grâce à des protéines de surface bactérienne. Parmi ces protéines, l’antigène 43 (Ag43) est sécrété par le système de sécrétion de Type V, sous-type a (T5aSS) et appartient à la famille des SAAT (self-associating autotransporters). Ces protéines modulaires, composées d’un peptide signal N-terminal clivable, d’un domaine passager central exposé à la surface et d’un translocateur C-terminal membranaire, peuvent s’auto-associer selon un mécanisme de Velcro moléculaire. A l’origine, l’Ag43 était distribué en deux sous-familles SF-I et SF-II mais des analyses phylogénétiques ont révélé pour la première fois qu’il est en réalité distribué en 6 classes différentes nommées C1 à C6, basées sur le réarrangement de différents sous-domaines à l’intérieur du domaine passager de la protéine. Quant à la prévalence et la distribution chez les bactéries, l’agn43 s’est avéré être présent essentiellement chez E. coli avec jusqu’à 5 copies du gène par génome, en différentes combinaisons de classes d’Ag43. Des analyses fonctionnelles des Ag43 C1 à Ag43 C4 ont démontré que des interactions homotypiques survenaient pour toutes les classes mais l’Ag43 C3 présentait des différences significatives de cinétique de sédimentation et d’autoagrégation. L’étude des interactions hétérotypiques entre Ag43 de différentes classes a démontré qu’elles ne se produisaient que dans de rares cas. Chez E. coli O157:H7, l’Ag43 C2 a été démontré comme contribuant à l’adhésion des bactéries aux collagènes I et III et à l’autoagrégation. De plus dans cette souche, ce phénotype s’est avéré être régulé par la méthylase Dam et le régulateur de transcription OxyR. Globalement, ces travaux apportent de nouvelles connaissances concernant la diversité de l’Ag43 et son rôle et sa régulation chez E. coli O157:H7. Ainsi, l’Ag43 pourrait être un important facteur de colonisation de la chaine alimentaire pour les EHEC et les autres E. coli diarrhéiques. / Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 are anthropozoonotic agents leading to haemorrhagic colitis and haemolytic-uremic syndrome (HUS). From the ruminants, their natural reservoir, EHEC can contaminate some foodstuffs and consequently infect humans. The presence of extracellular matrix (ECM) components along the food chain could contribute to the colonisation process. Numerous proteins can be present at the bacterial cell surface in EHEC, among them, antigen 43 (Ag43) is secreted by the Type V, subtype a, secretion system (T5aSS) and belongs to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. Ag43 was originally considered as distributed into two subfamilies, namely SF-I and SF-II but phylogenetic network analyses revealed for the first time that it actually distribute into six distinct classes, namely C1 to C6 based on the shuffling of different subdomains within the Ag43 passengers. Regarding prevalence and distribution in Bacteria, agn43 appeared to be essentially present in E. coli with up to 5 copies of the gene present in a single genome and in different combinations of Ag43 classes. Functional analyses of Ag43 C1 to Ag43 C4 revealed that homotypic interactions occurred for all Ag43 classes but Ag43 C3 induced significant differences in the sedimentation kinetics and autoaggregation state. In contrast, heterotypic interactions occurred in a very limited number of cases. In E. coli O157:H7, Ag43 C2 was shown to contribute to bacterial adhesion to collagens I and III and to the autoaggregation. Furthermore in this strain, the latter phenotype appeared to be regulated by the Dam methylase and the OxyR transcriptional regulator. Taken together, these results provide new insights of the diversity of Ag43 as well as its role and regulation in E. coli O157:H7. Thus, Ag43 may represent an important colonisation factor of the food chain by EHEC and other diarrheagenic E. coli.

Escherichia coli enterohemorrágicas (EHEC) que possuem a Ilha de Patogenicidade LEE são importantes patógenos humanos. A habilidade de adesão, invasão e perfil genético têm sido estudados, já que sorotipos que não possuem LEE tem sido isolados de pacientes com doença severa e intracelularmente. Das nove amostras relacionadas com doença, quatro (44.5%) apresentaram stx2+. Todas foram positivas para o gene lpfA e iha, e negativas para toxB. Três (33,3%) apresentaram saa e cinco hly. A maioria apresentou padrão de adesão difusa e invasão negativa em células HEp-2. Em CaCO-2 apresentaram aderência com variados graus de intensidade, e a maioria das amostras testadas apresentou invasão maior que 3,3%. Frente ao inibidor de polimerização de actina citocalasina D, houve significativa redução nos níveis de invasão, sugerindo que estas amostras utilizam um mecanismo da célula hospedeira para internalização, e que provavelmente, fatores de virulência como adesinas, favorecem a adesão das mesmas, compensando a ausência do LEE, e facilitando a instalação da infecção. / Enterohaemorrhagic Escherichia coli (EHEC) that possess the pathogenic island LEE are important human pathogens. The adhesion ability, invasion and genetic profile have been studied, since serotypes that do not possess LEE have been isolated from humans with severe disease and found intracellular. Nine strains related with SHU, four (44,5%) were stx2+. All strains were positive for IpfA and iha genes and negative for toxB. Three (33,3%) showed saa and five hly. The most strains showed a diffusely adhesion pattern and negative invasion in HEp-2 cells. It presented various degrees of adhesion, and the most tested strains showed invasion high than 3,3% in CaCO-2. That was a significant reduction of invasion in the presence of actin polymerization inhibitor Citochalasin D, suggesting that these strains use a host cell mechanism to invade, and probably virulence factors, like adhesins, favors this adhesion and compensate LEE absence, promoting the installation of infection.

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d’un syndrome hémolytique et urémique. Une fois arrivés dans l’intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d’attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d’effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l’expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l’expression du LEE. GadE active l’expression de gadX, et GadX réprime l’expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l’expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l’expression de gadE et de gadX. Le monoxyde d’azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l’expression du LEE et active celle de gadE et de gadX. Notre étude a permis d’identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l’expression de gadE et gadX et active l’expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l’expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l’expression de gadE et donc de gadX. Un modèle de régulation intégrant l’ensemble de ces résultats est proposé. D’autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d’hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l’expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC. / Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen causing hemorrhagic colitis and Hemolytic and Uremic Syndrome (HUS). After reaching the gut, EHEC adhere to the epithelial intestinal cells causing attachment/effacement lesions (A/E lesions). The locus of enterocyte effacement (LEE) encodes for a type three secretion system and several effector proteins required for A/E lesions. The LEE is composed of five main operons (LEE1-5). In this work we identified the molecular mechanisms of one of the regulatory cascades controlling LEE expression. Using qRT-PCR and chromatin immunoprécipitation we determined that GadE and GadX are two indirect repressors of LEE expression. GadE activates gadX expression, and GadX represses ler expression, the latter encoding the main activator of LEE2-5 operons. Moreover, GadE also represses LEE4 and LEE5 expression independently of Ler. In turn, Ler represses gadE and gadX expression. Nitric oxide (NO) is a crucial effector of the innate immune response, in part produced by intestinal epithelial cells. It has been shown previously that NO represses LEE and activates gadE and gadX expression. In this study we identified the key regulator responsible for these regulations: NsrR. NsrR represses indirectly gadE and gadX expression and activates LEE1, LEE4 and LEE5 expression by binding to their respective promoter. In the presence of NO, NsrR is inactivated. Thus, NO directly represses LEE expression by relieving NsrR binding to the LEE1, LEE4 and LEE5 promoters, and indirectly by activating gadE and gadX expression. A regulatory model is proposed based on these results.In addition, we identified and characterized a new phosphodiesterase which is specific for the most virulent EHEC strains: VmpA. By degrading c-di-GMPc, VmpA controls motility, biofilms formation, and probably LEE expression. It would also have a global effect on EHEC physiology.