L’étiologie exacte des maladies inflammatoires chroniques de l’intestin (MICI) reste actuellement méconnue. Mais un déséquilibre de la flore bactérienne, plus connu sous le nom de dysbiose et se traduisant par une augmentation de bactéries potentiellement pathogènes versus une diminution de bactéries bénéfiques, est démontré en permanence. De précédentes études ont permis de mettre en évidence la présence de souches pathogènes d’E. coli chez les patients atteints de la maladie de Crohn (MC). Ces souches appartiennent au pathovar Adherent Invasive E. coli (AIEC) et sont caractérisées par leur capacité à adhérer et à envahir les cellules épithéliales intestinales, à survivre et à se multiplier dans les macrophages en induisant une synthèse intense de TNF. La mise en évidence de ce pathovar a essentiellement été réalisée sur des biopsies de patients présentant une MC. Et bien que les mécanismes de pathogénicité et de virulence de la souche AIEC soient clairement déterminés, il n’existe pas d’études approfondies sur la prévalence des AIEC au niveau des matières fécales chez les patients atteints de MICI en comparaison à des individus sains. Ainsi, ce projet de thèse s’inscrit dans une meilleure compréhension de l’implication de ce pathovar AIEC dans les MICI au niveau luminal. Cette thèse cible différents points: prévalence et détection des AIEC, leur proportion relative par rapport à la flore E. coli totale, leur capacité d'invasion, leur phylogroupe ainsi que leur transmissibilité. A l’issue de ce travail, nous montrons que les AIEC sont retrouvés au niveau luminal chez les patients atteints de la MC mais également chez les patients présentant une rectocolite hémorragique, avec une détection des AIEC chez 33% et 2% respectivement. En outre, ces études ont permis de montrer une prévalence plus forte de ce pathovar dans les matières fécales d’individus sains (51%) en comparaison aux patients atteints de MICI. Et lorsque les AIEC sont présents, que ce soit chez les patients atteints de MICI et chez les témoins, ils représentent en moyenne 20 à 30% de la flore E. coli. Nous avons également pu montrer qu’il n’existe pas de différences significatives des scores d’invasion des isolats AIEC chez les patients atteints de MICI et chez les sujets sains. Certaines souches d’AIEC, isolées chez les patients atteints de MC et chez les sujets sains, ont été caractérisées génétiquement par la technique d’électrophorèse sur gel en champ pulsé. Sur ces souches, différents profils génétiques ont été obtenus attestant de la forte variabilité intra- et interindivuelle des AIEC. En conclusion, les AIEC, au vue de leur forte prévalence chez des sujets en bonne santé, seraient plutôt à reconsidérer comme des pathobionts ce qui définit un symbionte pouvant acquérir des propriétés virulentes chez un hôte prédisposé génétiquement en raison de facteurs environnementaux et/ou diététiques et ainsi favoriser l’inflammation intestinale. / Many studies have reported an imbalance of bacterial flora in patients with inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), defined as a dysbiosis, and resulting in an increase in potentially pathogenic bacteria versus a decrease in beneficial bacteria. Previous studies have highlighted the presence of pathogenic strains of E. coli in patients with CD. These strains belong to the pathovar Adherent Invasive E. coli (AIEC) and are characterized by their ability to adhere and invade intestinal epithelial cells, to survive and to multiply in macrophages by inducing an intense synthesis of TNF. In recent years, many studies established a link between AIEC pathovar and CD. Many of these studies have been performed on biopsies of patients with CD. And although the mechanisms of pathogenicity and virulence of the AIEC strain are clearly determined, there are no in-depth studies on the prevalence of AIEC in feces in IBD patients in comparison to healthy individuals. Thus, the goal of this thesis project is to better understand the involvement of AIEC pathovars in IBD at the luminal level. This thesis is based more precisely on the study of the prevalence of AIEC in feces of patients with IBD in comparison to healthy subjects, targeting different points: prevalence and detection of AIEC, their relative proportion compared to total E. coli flora, their invasion capacity, their phylogroup as well as their transmissibility. AIEC are found at luminal level in patients with CD but also in patients with UC, with detection of AIEC in 33% and 2% respectively. In addition, a higher prevalence of these pathovar is present in the feces of healthy individuals (51%) compared to patients with IBD. And when AIEC are present, both in IBD patients and in controls, they represent on average 20 to 30% of the E. coli flora. We have also been able to show that there are no significant differences in AIEC invasion scores in patients with IBD and in healthy subjects. Some AIEC strains, isolated in patients with CD and in healthy subjects, have been genetically characterized by pulsed-field gel electrophoresis. Different genetic profiles have been obtained attesting the high intra- and interindividual variability of AIEC strains. In conclusion, because of their high prevalence in healthy individuals, AIEC should be reconsidered as pathobionts, which defines a symbiont acquiring virulent properties in a genetically predisposed host due to environmental and / or dietary factors and thus promoting intestinal inflammation.

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie est multifactorielle. Les lésions iléales des patients atteints de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC («Adherent-Invasive E. coli»). Ces souches sont capables d'adhérer et d'envahir les cellules épithéliales intestinales, et ont la capacité de survivre et de se multiplier fortement en macrophages en induisant une synthèse intense de TNF-α. L'objectif de ce travail s'inscrit dans la compréhension des mécanismes permettant aux bactéries AIEC de coloniser la muqueuse intestinale et d'induire les stades précoces de la pathologie. Une précédente étude menée au laboratoire avait permis de mettre en évidence l'importance de l'activation de la voie de régulation dépendante du facteur bactérien sigma alternatif RpoE (ou σE) dans le processus d'adhésion et d'invasion des cellules épithéliales intestinales par la souche AIEC de référence LF82 via l'expression des pili de type 1 et des flagelles. En continuité de ces travaux, nous montrons que l'activation de la voie de signalisation dépendante du facteur σE est également primordiale pour la capacité des souches AIEC à former des biofilms, et une analyse bioinformatique ayant pour but d'identifier les gènes régulés par σE a montré que l’opéron waaWVL, impliqué dans la biosynthèse du lipopolysaccharide, est primordial pour la formation de biofilm par les souches AIEC. De plus, nous avons mis en évidence que les long polar fimbriae (LPF) sont impliqués dans le ciblage de l'épithélium associé aux plaques de Peyer par les bactéries AIEC, et ceci en leur permettant de cibler spécifiquement les cellules M. L'inactivation du gène Nod2, gène de susceptibilité à la MC, conduit à une augmentation du nombre de plaques de Peyer ainsi que des cellules M à leur surface, indiquant que les bactéries AIEC pourraient tirer avantage d'une susceptibilité génétique pour cibler les plaques de Peyer. / 2026/5000Crohn's disease (CD) is a chronic inflammatory condition of the gastrointestinal tract with a multifactorial etiology. The ileal lesions of MC patients are abnormally colonized by pathogenic strains of Escherichia coli belonging to the AIEC ("Adherent-invasive E. coli") pathovar. These strains are able to adhere and invade intestinal epithelial cells, and have the ability to survive and multiply strongly in macrophages by inducing intense TNF-α synthesis. The aim of this work is to understand the mechanisms by which AIEC bacteria colonize the intestinal mucosa and induce early stages of the pathology. A previous study carried out in the laboratory had made it possible to highlight the importance of the activation of the regulatory pathway dependent on the bacterial factor sigma alternative RpoE (or σE) in the process of adhesion and invasion of intestinal epithelial cells by the AIEC reference strain LF82 via expression of type 1 pili and flagella. As a continuation of this work, we show that the activation of the σE-dependent signaling pathway is also essential for the ability of AIEC strains to form biofilms, and a bioinformatic analysis aimed at identifying σE-regulated genes. showed that the waaWVL operon, which is involved in the biosynthesis of lipopolysaccharide, is essential for biofilm formation by AIEC strains. In addition, we have shown that long polar fimbriae (LPF) are involved in the targeting of Peyer's plaque-associated epithelium by AIEC bacteria, allowing them to specifically target M cells. Inactivation of the MC susceptibility gene, Nod2, leads to an increase in the number of Peyer's patches as well as M cells on their surface, indicating that AIEC bacteria could benefit from genetic susceptibility to target Peyer's patches.

Mon travail a porté sur la caractérisation des effecteurs toxiques et protéines d’immunité du T6SS Sci-1 d’Escherichia coli Entero-agrégatif, éléments de la lutte inter-bactérienne. Nous avons identifié en outre Tle1, un effecteur de toxine codé par ce groupe et montré que Tle1 possède des activités de phospholipase A1 et A2 requises pour détruire la cellule proie dans la compétition interbactérienne. L'auto-protection de la cellule attaquante est assurée par une lipoprotéine de membrane externe, Tli1, qui lie Tle1 dans un rapport stoechiométrique 1: 1 avec une affinité nanomolaire et inhibe son activité phospholipase. Il a été prédit que la protéine 435 provenant à partir d'un groupe de gènes T6SS1 de l'agent pathogène AIEC LF82 est une phospholipase de la famille d'effecteurs Tle3 avec une activité PLA1. Sa toxicité peut être neutralisée par la protéine d'immunité cognate 434 qui est un Tli3 putatif, en formant le complexe de protéine Tle3 - Tli3. Les deux protéines séparées et leur complexe ont ensuite été appelées protéines complexes Tle3AIEC, Tli3AIEC et Tle3AIEC - Tli3AIEC, respectivement. Afin d'étudier plus en détail le mécanisme de Tle3-AIEC et de Tli3-AIEC, nous avons réalisé l'expression, la purification, la caractérisation, la cristallisation des deux protéines et des études cristallographiques de rayons X préliminaires du complexe Tle3-AIEC/Tli3-AIEC afin de comprendre comment la protéine Tle3-AIEC reconnaît et se lie à son effecteur apparenté Tli3-AIEC et inhibe son activité. Les données préliminaires de diffraction des rayons X ont été recueillies à partir de cristaux Tle3AIEC-SeMet/Tli3AIEC à une résolution de 3,8 Å. / Here, we analyzed the Entero-aggregative Escherichia coli Sci-1 T6SS toxin effectors. We identified Tle1, a toxin effector encoded by this cluster and show that Tle1 possesses phospholipase A1 and A2 activities required for the inter-bacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity.The protein 435 from the pathogen AIEC LF82 has been predicted to be a phospholipase of the Tle3 effector family with PLA1 activity from a T6SS1 gene cluster. Its toxicity can be neutralized by the cognate immunity protein 434 that is a putative Tli3, by forming Tle3 - Tli3 protein complex. The two separated proteins and their complex were then called Tle3AIEC, Tli3AIEC and Tle3AIEC - Tli3AIEC complex proteins, respectively. In order to further investigate the related mechanism of Tle3AIEC and Tli3AIEC, we performed expression, purification, characterization, crystallization of the two proteins and preliminary X-ray crystallographic studies of the Tle3AIEC - Tli3AIEC complex in order to understand how Tle3AIEC protein recognizes and binds to its cognate Tli3AIEC effector and inhibits its activity. X-ray diffraction data were collected from selenomethionine-derivatize Tle3AIEC SeMet - Tli3AIEC crystals to a resolution of 3.8 Å.

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l’étiologie est multifactorielle. Les lésions intestinales des patients atteints de MC sont anormalement colonisées par des souches pathogènes d’Escherichia coli appartenant au pathovar AIEC pour «Adherent-Invasive Escherichia coli ». Ces souches sont capables d’adhérer et d’envahir les cellules épithéliales intestinales, et ont la capacité de survivre et de se multiplier en macrophages en induisant une synthèse intense de cytokines pro-inflammatoires. Les AIEC pourraient ainsi être impliquées dans l’induction et/ou l’entretien de l’état inflammatoire de la muqueuse intestinale.L’objectif de ce travail est d’identifier les déterminants bactériens des AIEC qui vont intervenir dans les étapes précoces de l’implantation des AIEC au niveau intestinal et de définir quel est le rôle des facteurs micro-environnementaux de l’hôte dans cette implantation.Nous montrons que l’AIEC LF82 possède une activité mucinolytique qui est portée par le gène vat-AIEC et qui favorise la traversée du mucus et la colonisation intestinale. Nous avons retrouvé ce gène chez 42% des souches AIEC isolées de patients atteints de MC, et chez 97% des souches AIEC appartenant au phylogroupe B2. Par ailleurs, nous avons montré que les sels biliaires augmentent l’expression de cette mucinase.Nous avons ensuite étudié l’influence des sels biliaires sur l’expression globale des gènes de la souche LF82. Les sels biliaires modifient profondément le métabolisme de la souche, induisant une diminution globale des voies de biosynthèse (protéines, lipides) et une augmentation des voies de dégradation (alcools, acides carboxyliques, polyamines, …). L’étude du catabolisme de l’éthanolamine et du propanediol indique que les AIEC pourraient se servir de ces substrats pour s’implanter au sein de la flore iléale. De plus, les analyses transcriptomiques révèlent que les sels biliaires augmentent l’expression de gènes codant des facteurs de virulence comme l’invasine IbeA, les systèmes de sécrétion de type VI et la yersiniabactine. Nous montrons également qu’ils favorisent la formation de biofilm chez les souches AIEC.Ces données indiquent que les sels biliaires constituent un signal permettant à la souche AIEC LF82 de mettre en place différentes voies métaboliques et déterminants bactériens nécessaires à son implantation au niveau intestinal.Mots-clé : Escherichia coli, maladie de Crohn, mucines, serine protéase, mucinase, AIEC, / The etiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Ileal mucosa of CD patients is often abnormally colonized by adherent-invasive Escherichia coli (AIEC). These strains isolated from the intestinal mucosa of CD patients are able to adhere to intestinal epithelial cells (IECs). This adhesion to IECs promotes the invasion of cells by the bacteria. Furthermore, the invasive ability of AIEC strains allows bacteria to translocate across the human intestinal epithelium, move into the deep tissues and activate immune cells continuously upon arrival. Thus AIEC could be involved in the inflammatory state of the intestinal mucous membrane. The aim of this study was to identify components of AIEC virulence, which might favor their implantation in the gut of CD patients and to define the role of several chemical factors from the ileal environment. Here, we reported a protease called Vat-AIEC from AIEC which favors the penetration of AIEC through the mucus layer and enhances gut colonization. The screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Besides, Vat-AIEC transcription was increased with bile salts from the ileum environment. Then a global RNA sequencing (RNA-seq) of E. coli LF82 has been used to observe the impact of bile salts on the expression of bacterial genes. The results demonstrate the explosive effect of bile salts with a dysregulation of about 40% of the genome, with a global upregulation of genes involved in degradation and downregulation of those implicated in several biosynthesis. Our results show that LF82 use ethanolamine as a nitrogen source and propane diol as a carbon source, which can favor their colonization in the gut compared to the other bacteria. We also studied virulence genes expression in the presence of bile salts. They increase the expression of several virulence factors like the IbeA invasion, the type 6 secretion systems and the yersiniabactin. Furthermore, we noticed an increased expression of genes implicated in biofilm formation. These results improve the understanding of the global regulatory network in the presence of bile salts and thus of AIEC implantation in the human gut of CD patients.

<p>Crohn’s Disease is a chronic inflammatory bowel disease characterized by an overzealous immune response to a microbial trigger in genetically susceptible individuals. Although this microbial trigger is unknown, <em>Escherichia coli</em> with adherent and invasive properties (Adherent-Invasive <em>Escherichia coli</em>, AIEC) is preferentially enriched in a proportion of Crohn’s Disease patients. AIEC can adhere to and invade intestinal epithelial cells and replicate intracellularly within epithelial cells and macrophages <em>in vitro</em>. One important colonization factor expressed by AIEC is the type 1 fimbrial adhesin protein FimH. FimH mediates colonization of CEABAC10 transgenic mice and can bind several host cell receptors including the macrophage receptor CD48 <em>in vitro</em> indicating a potential role for FimH in macrophage interaction. However, it was not known whether FimH contributed to phagocytosis of AIEC or colonization of wild-type mice. Here we show that FimH enhances early intracellular AIEC levels <em>in vitro</em> and colonization <em>in vivo</em>. We found that deletion of <em>fimH</em> may reduce intracellular AIEC burden at 2 hours post-infection and that this effect was modulated by bacteria opsonisation. Using a competitive index assay, we show that a Δ<em>fimH</em> mutant is unable to chronically colonize CD-1 mice at the same levels as the parental strain. Our results demonstrate that FimH is an important AIEC colonization factor and may increase interaction with macrophages. Identifying factors such as FimH which contribute to colonization and persistence will further our understanding of AIEC survival strategies within the host. Development of therapeutics targeting FimH may provide a means to reduce harmful bacteria overgrowth particularly after surgical intervention.</p> / Master of Science (MSc)

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

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Inflammatory bowel disease (IBD), comprises Crohn’s disease (CD) and ulcerative colitis (UC), and is a chronic relapsing inflammation of gastrointestinal tract without any known cause or cure. Currently, it is accepted that IBD is a result of a dysfunctional immune response to commensal bacteria in a genetically susceptible host, and that environmental factors can trigger the onset or reactivation of the disease. This thesis considers the possibility of a specific pathogenic agent as well as an imbalance in the composition of the normal microflora in the pathogenesis of IBD. Gut biopsy tissues were taken from a population-based case-control tissue bank held at the University of Manitoba. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were employed to assess the diversity of gut microbiota. The phylogenetic, virulence and biochemical characteristics of Escherichia coli isolated from IBD biopsies were examined using multi-locus sequence typing (MLST), DNA microarray technology and API 20E system. Utilizing ARISA and T-RFLP, a remarkable increase in the order of unclassified Clostridia was detected in inflamed tissues, particularly in CD patients (P < 0.05). Moreover, species richness and diversity were the highest in non-inflamed IBD biopsies. Culture-based quantification detected a significantly higher number of E. coli in IBD tissues (P < 0.05). Phylogenetic analysis revealed the tendency of E. coli isolated from IBD patients to be grouped into separate clonal clusters based on their allelic profiles (P = 0.02). A link was detected between uropathogenic E. coli (UPEC) CFT073 and strains isolated from IBD, with regards to gene distribution and virulence, using microarray technology. Amino acid substitutions N91S and S99N in FimH, the adhesive subunit of E. coli type I fimbria, were significantly associated to IBD (P < 0.05). This study demonstrated an increase in the microbial diversity of non-inflamed IBD tissues and suggested a recruitment phase of bacterial adherence and colonization, before the inflammation sets in. Furthermore, E. coli isolated from IBD tissues were distinct from commensal strains in both clonal and virulence characteristics and shared remarkable traits with extraintestinal pathogenic E. coli. Features involved in bacterial adhesion to epithelial cells may hold the key to E. coli pathogenesis in IBD.

Inflammatory bowel disease (IBD), comprises Crohn’s disease (CD) and ulcerative colitis (UC), and is a chronic relapsing inflammation of gastrointestinal tract without any known cause or cure. Currently, it is accepted that IBD is a result of a dysfunctional immune response to commensal bacteria in a genetically susceptible host, and that environmental factors can trigger the onset or reactivation of the disease. This thesis considers the possibility of a specific pathogenic agent as well as an imbalance in the composition of the normal microflora in the pathogenesis of IBD. Gut biopsy tissues were taken from a population-based case-control tissue bank held at the University of Manitoba. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were employed to assess the diversity of gut microbiota. The phylogenetic, virulence and biochemical characteristics of Escherichia coli isolated from IBD biopsies were examined using multi-locus sequence typing (MLST), DNA microarray technology and API 20E system. Utilizing ARISA and T-RFLP, a remarkable increase in the order of unclassified Clostridia was detected in inflamed tissues, particularly in CD patients (P < 0.05). Moreover, species richness and diversity were the highest in non-inflamed IBD biopsies. Culture-based quantification detected a significantly higher number of E. coli in IBD tissues (P < 0.05). Phylogenetic analysis revealed the tendency of E. coli isolated from IBD patients to be grouped into separate clonal clusters based on their allelic profiles (P = 0.02). A link was detected between uropathogenic E. coli (UPEC) CFT073 and strains isolated from IBD, with regards to gene distribution and virulence, using microarray technology. Amino acid substitutions N91S and S99N in FimH, the adhesive subunit of E. coli type I fimbria, were significantly associated to IBD (P < 0.05). This study demonstrated an increase in the microbial diversity of non-inflamed IBD tissues and suggested a recruitment phase of bacterial adherence and colonization, before the inflammation sets in. Furthermore, E. coli isolated from IBD tissues were distinct from commensal strains in both clonal and virulence characteristics and shared remarkable traits with extraintestinal pathogenic E. coli. Features involved in bacterial adhesion to epithelial cells may hold the key to E. coli pathogenesis in IBD.