Project description:The role of propionate-induced rearrangement of membrane proteins in the formation of the virulent phenotype of Crohn's Disease-associated adherent-invasive Escherichia coli. Adhesive-invasive Escherichia coli (AIEC) were first isolated from the ileal mucosa of a patient with Crohn's disease (CD) which is a severe chronic immune-mediated granulomatous inflammatory disease of the gastrointestinal tract. It turned out that they are able to successfully penetrate the mucin layer, overcome the epithelial barrier, and also survive and multiply inside macrophage. Bacteria with such properties were assigned to a special group of pathobiont adhesive-invasive E. coli. AIEC activity is accompanied by the release of pro-inflammatory cytokines, i.e. surviving and multiplying inside macrophages, they enhance the inflammatory process. The role of AIEC in the onset or chronicity of CD is not well-defined. However, it has been proposed that these bacteria could trigger the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells, and then, due to their survival within macrophages, they could stimulate chronic inflammation and granuloma developmen. Pathogens use a variety of mechanisms, including the induction of inflammation, the direct or indirect destruction of commensal species, and the use of alternative carbon sources to survive. AIEC are shown to be able to utilize ethanolamine and propanediol, which are formed during the catabolism of phospholipids, fucose or rhamnose, propionate and other metabolites. Metabolic plasticity is thought to allow AIEC to act as an opportunistic pathogen in conditions of intestinal inflammation. We have previously shown that passage of AIEC from a CD patient (CD isolate) on M9 minimal medium supplemented with sodium propionate (PA) as a carbon source leads to a strong increase, and passage on M9 medium supplemented with glucose, on the contrary, leads to a significant decrease in adhesive-invasive properties and ability to survive in macrophages. We were able to compare the isogenic CD isolate in two states: virulent with high adhesive-invasive activity and ability to survive in macrophages, and non-virulent, when these properties are lost. In contrast to the CD isolate, passage of the laboratory strain K12 Mg1655 on the M9 medium supplemented with PA did not cause a similar effect. We performed a comparative proteomic analysis of membrane fractions isolated from ZvL2-PA and ZvL2-GLU using LC-MS. The laboratory strain K12 Mg1655 was used as a control.

Project description:The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.

Project description:Crohn’s disease (CD) is a severe chronic immune-mediated granulomatous inflammatory disease of the gastrointestinal tract. Its pathogenesis mechanisms remain obscure. One of the factors of CD pathogenesis is reduced diversity of gut microbiota which is accompanied by the characteristic increase of representation of Escherichia coli strains, several of which are able to penetrate the mucin layer, adhere to the epithelial cells, cross the epithelial barrier, and colonize macrophages. During the internalization, these E. coli strains can modulate macrophages’ defensive functions in a way that allows them to survive in a presence of such negative factors as low pH, oxidative stress, proteolytic enzymes and antimicrobial solutions. These strains were collectively assigned to the adhesive–invasive group of E. coli (AIEC). Earlier we sequenced 28 E. coli isolates from 10 patients with CD [Rakitina DV et al. Genome analysis of E. coli isolated from Crohn disease patients. BMC Genomics. 2017, 18(1):544. doi: 10.1186/s12864-017-3917-x] It has been demonstrated that the majority of enteropathogens feature specific metabolic pathways allowing them to use alternative carbon sources which leads to survivability and enhaced competitiveness in the host organism. Previously, we demonstrated that virulent properties of CD isolates are dependent on the carbon source: prolonged growth on propionate containing medium stimulates virulent properties while prolonged growth on glucose reduces these properties to levels indistinguishable from laboratory strain K-12 MG1655 [Pobeguts OV et al. Propionate Induces Virulent Properties of Crohn’s Disease-Associated Escherichia coli. Frontiers in Microbiology. 2020, 11:1460. doi: 10.3389/fmicb.2020.01460]. Thus, cultivation of E. coli on different growth mediums: M9 supplied with propionate and glucose made it possible to distinguish two states of each CD isolate: active (aAIEC) and inactive (iAIEC). In order to identify the major changes that occur upon change of carbon source, we carried out LC–MS-based proteomic analysis of aAIEC and iAIEC states of three CD isolates of E. coli ZvL2 (RCE07), BruB2 (RCE03) and К5 (RCE06) grown on glucose and propionate.

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E. coli Pathobionts and Crohn’s Disease: Varied genetic paths leading to similar phenotypes

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