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:The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic used for chronic multidrug resistant infections infections, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can augment colorectal cancer (CRC) through DNA double stranded breaks and interstrand crosslinks. While the structure and biosynthesis of colibactin has been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by pathogenic E. coli are largely unexplored. This research highlights the regulation of the colibactin-producing biosynthetic gene cluster under polymyxin stress. Using a multi-omic approach, we have identified that polymyxin stress enhances the abundance of colibactin biosynthesis proteins (Clb’s) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC: NC101, the probiotic strain: E. coli Nissle 1917, and the antibiotic testing strain: E. coli ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb’s by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with heightened tolerance for polymyxin antibiotics induced greater DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation, the microbial response to antibiotics in the gut, and the ability of seemingly innocuous commensal microbes to induce host disease.

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Escherichia coli LF82

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