A high prevalence of invasive Escherichia coli strains, designated as adherent-invasive E. coli (AIEC), has been frequently observed in the ileal mucosa of Crohn’s disease patients. Preventing the colonization of ileal mucosa by AIEC could be a strategy to limit recurrence of CD. Based on solid preliminary data, our hypothesis is that deregulated Histone deacetylase (HDAC) expression/activity in intestinal epithelial cells of CD patients, induced by environmental factors such as western diet, leads to altered gene expression facilitating Enterobacteria colonization. The main objective of this proposal is to highlight the molecular mechanisms leading to AIEC colonization in CD patients to propose new therapeutic targets to limit/prevent AIEC intestinal colonization. To reach this objective, we aim (1) to study the role of HDAC1 and HDAC5 in the control of AIEC intestinal colonization and in inflammatory response, (2) to identify HDAC1- and HDAC5-regulated genes involved in the process of AIEC invasion, and (3) to identify the molecular mechanisms favoring AIEC colonization of intestinal mucosa in mice fed a western diet with a focus on the role of HDAC. This work will be carried out in various cellular models and in genetically modified mice models. In addition, the results will be validated in a cohort of 180 patients for which transcriptomics data and intestinal colonization by Enterobacteriaceae / AIEC levels are available. The identification of host factors favoring Enterobacteria and more specifically AIEC encroachment to intestinal mucosa should lead to the proposal of new alternative strategies to prevent AIEC colonization in CD patients.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD), are contemporary conditions of industrialized societies. The global evolution of IBD implicates a potential sequence in the pathogenesis of IBD: Westernization alters environmental exposure that influences the microbiome that incites IBD in genetically susceptible individuals. Understanding the elements of this sequence is critical in order to halt the progression of IBD. The lifetime risk of developing IBD has been calculated to be 1%. All ages can be affected but CD mostly begins in young adulthood. It has been estimated that over 2 million people are affected with IBD in Europe. In newly industrialized countries, the incidence of IBD especially that of CD has been increasing. Consequently, slowing down the incidence of CD is paramount. Experimental and observational data suggest that intestinal inflammation in CD arises from abnormal immune response to intestinal microbiota in genetically susceptible individuals. Genes that regulate innate immune response, intestinal barrier function and bacteria killing of intracellular pathogens have been associated with an increased risk for developing CD in Caucasian populations. The search for specific pathogens in CD has identified in the intestinal mucosa of patients several candidates. One with much supporting evidence is the adherent invasive Escherichia coli (AIEC). Since its discovery, several groups have reported a higher prevalence of AIEC in CD patients compared to healthy subjects and confirmed their pro-inflammatory potential. A growing body of work indicates that different host environments can select such AIEC pathobiont. AIEC colonization in mice leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD. Furthermore the presence of AIEC in mucosa of CD patients at diagnosis suggests they may play a role in early stages of disease onset. Much of the work on this organism, and its potential role in CD, has been undertaken in areas of high CD incidence (Europe, North America and Australia). It is currently unclear if the changes in such putative pathogens organisms identified in Western populations are more widespread in IBD patients in regions with increasing IBD incidence, especially in Asia. Information is also lacking about the degree of genetic variation between the bacteria assigned to these taxonomic groups from different ethnic and geographical regions. The significance of AIEC pathobiont in the pathogenesis of CD worldwide is currently unknown. For this organism to be considered truly implicated in CD, its presence in patients in other parts of the world including countries with high disease incidence and countries with increasing incidence would need to be demonstrated. In the past few decades, the incidence of CD is increasing in developing countries. In 2011, the first and largest population-based study was conducted by our group consisting of 13 countries in Asia-Pacific. We showed that Hong Kong is amongst the top three countries in Asia with the highest incidence of CD. The incidence of CD in Hong Kong has increased by 30-fold in the past two decades. In addition, CD incidence varies between rural and urban area within China, with a higher incidence in urban areas, likely as the result of the influence of environmental factors including diet. The main objective of this study is to assess the prevalence, characteristics and genetics of pathogenic AIEC in ileal CD across two different distinct populations and geography (Caucasians in France and Chinese in Hong-Kong) and to delineate therapeutics targeting AIEC in individuals with Crohn’s disease. These data will provide an improved understanding on whether AIEC is a risk factor for Crohn’s disease globally, the pathogenicity of AIEC and possible treatment based on AIEC eradication in Crohn’s disease which can lead to a personalized therapeutic case based approach in patients with Crohn’s disease.

Accumulating evidences revealed that disturbances of the host-microbiota relationship can results in microbiota encroachment which associates with the promotion of chronic intestinal inflammation. For example, we observed that consumption of dietary emulsifiers leads to alterations in microbiota that associate with increased colitis severity and metabolic deregulations in mice. Emulsifier-induced microbiota alterations are necessary and sufficient to promote inflammation, and we more recently demonstrated that specific microbiota members, yet to be identified, are required to drive the detrimental effects of emulsifiers. The EMULBIONT project will built upon these previous observations to identify bacteria, as well as molecular mechanisms, driving emulsifier-induced chronic inflammation. We anticipate that completion of this project will provide important knowledge on mechanisms by which select food additives can shape a pro-inflammatory microbiota, with the ultimate goal to provide microbiota-based personalized nutrition.

The intestinal microbiota is central in the antibiotic resistance phenomenon. Nonetheless, the impact of antibiotics on the intestinal microbiota remains poorly described, mainly because of the lack of phenotypic characterization of the resistance mechanisms of intestinal bacteria. Here, we propose to study the impact of various beta-lactams regimens (ceftriaxone, piperacillin-tazobactam and ceftazidime-avibactam, with or without a colon-delivered charcoal-based adsorbent) the intestinal microbiota of healthy volunteers, leveraging fecal antibiotic concentrations, state-of-the-art metagenomic analysis (combining short and long reads), phenotypic characterization, in vitro evolutionary models and longitudinal data analysis. Altogether, BLA-IMPACT will help in bridging phenotypic and metagenomic data that are mandatory to understand how the intestinal microbiota evolved under antibiotic exposure.

The Type VI secretion system (T6SS) is a macromolecular machine found in many Gram negative bacteria, including gut pathogens and commensals. T6SS is involved in interbacterial competition and virulence, through the secretion of different anti-prokaryotic, anti-eukaryotic or even transkingdom effectors (phospholipases, DNases). T6SS is thus directly involved in virulence, and/or can help to clear and colonize niches by killing competitors. The competitive advantage provided by T6SS on intra and inter-species bacterial interaction in the gut and thus its impact on the composition of the human microbiota and the pathogenesis outcome was recently highlighted. The T6SS consists of a membrane complex anchored in the bacterial envelope that recruits a platform assembly for a contractile tail structure. The tail is a tube wrapped by a sheath and topped by a needle spike. Upon contact, the contraction of the sheath propels the tube and the spike toward target cells. Toxic effectors interacting with the spike are delivered in bacteria and/or eukaryotic cells. Adherent-Invasive Escherichia coli (AIEC) are pathogenic E. coli strains frequently isolated from patients with ileal Crohn’s disease (CD), an inflammatory bowel disease. Numerous studies have confirmed mucosal association of invasive E. coli (between 21 to 63% of CD patients) in various cohorts of CD patients. AIEC strains are able to adhere to and invade intestinal epithelial cells, to survive and replicate within macrophages and colonize ileal gut mucosa in CD patients. Genome analysis of AIEC reference strain reveal the presence of pathogenic islands encoding T6SSs, but their contribution in the virulence of AIEC is not known. Our working hypothesis is that AIEC bacteria may use their T6SS and corresponding effector toxins to favour their colonization in the gut, either by direct elimination of their competitors or through the establishment of a favorable niche involving a shift towards a colitogenic microbiota and/or the induction of a low-grade mucosal inflammation. This proposal brings together the complementary expertise of two partners, at the molecular microbiology, cell biology and physiopathology interface. We will use a comprehensive approach of molecular biology, biochemistry, cell biology and animal models to determine the mode of action and delivery, as well as the contribution of AIEC T6SS effectors in AIEC pathogenicity. We will focus our work on the most prevalent T6SS and its corresponding effectors, two putative membrane-targeting toxins. We will dissect the molecular mechanisms underlying the secretion/translocation of AIEC effectors. In parallel, understanding the mode of action and delivery of Tle1phospholipase toxin by enteroaggregative E. coli, a model that is well established in Partner 1 laboratory, will guide us to achieve these goals. We will identify putative partners required for toxin activation in the target cell by fusing the toxins to the biotin ligase and by identifying biotinylated partners. We will test the participation of T6SS and of each effector in anti-bacterial and anti-host activities. We will determine effector activity, mode of action and consequences on the target cell. To decipher the role played by T6SS in dysbiosis-induced by AIEC, we will use several in vivo animal models to compare the impact of AIEC strains or their isogenic effector- or T6SS-invalidated mutant on gut microbiota. In vivo mouse models will include innovative mouse models with controlled microbiota. We will also analyse gut microbiota composition of CD patients colonized or not with T6SS+ or T6SS- AIEC. We expect to better understand the contribution of T6SS to AIEC pathogenicity. This project will clarify whether and how AIEC T6SS impact intestinal microbiota and physiology of gut mucosa. Better understanding of T6SS role in gut colonization will allow elaboration of new strategies to counteract and/or prevent AIEC implantation in the gut of CD patients.