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.