Escherichia coli (E. coli) are a diverse species of bacteria that reside, often harmoniously and beneficially, in the gastrointestinal tracts of humans and other mammals. However, some strains are associated with serious intestinal and extra-intestinal disease and are considered pathogens. The main differences between strains of these different E. coli pathotypes can be explained by the acquisition of genetic information introduced by mobile genetic elements, in particular bacteriophage. In enterohaemorrhagic E. coli (EHEC) O157:H7 strain EDL933, a pathotype of E. coli containing prophage-encoded Shiga toxins associated with severe gastrointestinal and systemic disease in humans, these horizontally acquired elements have been termed O-islands (OIs) and include both fully functional and cryptic prophages. The overall aim of this research was to try and determine what these OIs are actually doing for the bacteria. Systems pertinent in the life cycle and virulence of this pathogen were therefore investigated by phenotypically screening a large library of OI deletions in EHEC strain TUV93-0, a Shiga toxin-negative derivative strain of EDL933, and then comparing these with the parent strain. These analyses highlighted a subset of OIs with the potential to regulate motility and type III secretion (T3S), the latter being an essential colonisation factor for EHEC that is encoded by the locus of enterocyte effacement (LEE). Deletion of OI-51, a 14.93 Kb cryptic prophage designated as CP-933C, significantly reduced persistence of faecal shedding in sheep and levels of T3S expression in vitro. Cloning and complementation together with targeted allelic replacements in OI-51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the sequenced E. coli O157:H7 strain Sakai that is present but not annotated in the EDL933 sequence. Functionally important residues of ECs1581 were identified by site-directed mutagenesis based on phenotypic variants present in strains from different E. coli pathotypes, including strains not harbouring a LEE-encoded T3S system. This regulator was subsequently termed RgdR based on a motif demonstrated to be important for stimulation of gene expression from LEE1. Purified RgdR protein was able to form multiple complexes on a PCR generated LEE1 promoter fragment, and activation of this operon appeared to require this DNA binding capacity as a non-T3S inducing variant was unable to bind this same LEE1 promoter fragment. RgdR did not directly activate LEE1 transcription in vitro, nor did it activate transcription by relieving H-NS repression as proposed for the global regulator Ler (LEE-encoded regulator). However, RgdR activation did require a wild type LEE1 promoter and the Ler auto-induction cycle to induce LEE2-5 expression and T3S. RgdR was able to increase binding to Congo red and was capable of repressing bacterial motility. Further analyses demonstrated that RgdR did not regulate T3S and cell motility via GrlA (global regulator of LEE activator) and QseC (quorum sensing E. coli regulator C), two established regulators in E. coli that control LEE gene expression and motility in conjunction with their partners, GrlR (global regulator of LEE repressor) and QseB (quorum sensing E. coli regulator B) respectively. RgdR is therefore identified as a novel regulator able to co-ordinate T3S and motility expression. This research has identified OI-51 as being important for EHEC O157:H7 colonisation in sheep and has identified a completely new family of small bacterial regulators that control surface factor expression in E. coli.

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 EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine). / EHEC O157:H7 are an emerging zoonotic food- and water-borne hazard highly pathogenic to humans and associated with diseases ranging from acute gastroenteritis to hemolytic uremic syndrome. From their commensal carriage by farm animals to human targets, EHEC pass through a crucial step of persistence in the open environment. Microbial endosymbiosis is one strategy used by pathogenic bacteria to survive in aquatic environments. Amoebae species are free-living protozoa found in diverse environmental habitats and known to host several water-borne pathogens. Thus amoebae could contribute to transmission of EHEC to humans. The first part of my PhD project was focused on interaction of the free-living amoebae Acanthamoeba castellanii with EHEC. The results showed that the presence of amoeba extends the persistence of EHEC that survived phagocytosis by amoebae. This demonstrates the real potential of amoebae to harbourd EHEC that may contribute to their transmission. However, absence of shiga toxins enhanced the intra-amoeba survival. Moreover, EHEC had a toxic and lethal effect on amoebae partially due to shiga toxins. The involvement of shiga toxins in the intracellular survival and mortality of amoebae suggests the value of using amoebae as a model of host/pathogen interactions to study the pathogenicity of EHEC. During their transmission cycle, EHEC encounter limitation inorganic phosphate (Pi) in the environment. Using jointly the PhoB-R two-component system (TCS) and the Pst (Pi specific transport) system, EHEC detect and respond to this Pi limitation by activating the Pho regulon. The interplay between the EHEC virulence, the Pho-Pst and/or the environmental Pi remains unknown. The second part of my project explored the role of Pho regulon (responding to Pi-limitation stress) in the virulence of EHEC. Transcriptomic analysis showed that EHEC has evolved a sophisticated response to Pi deficiency involving not only biochemical strategies that are likely critical to its survival, but also coordinating its virulence response. In these conditions, the regulator PhoB regulates directly the expression of LEE and Stx2 genes. This is confirmed by an increase in EspB secretion and Stx2 production and secretion in low Pi conditions. Moreover, the activation of Pho regulon increases biofilm formation and reduces motility in EHEC. This correlated with the induction of genes regulating curli production and repression of indole production pathway and the flagellum and PGA biosynthesis.

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.