Imprinting of the neonatal gut epithelium by flagellated commensal bacteria

Aim: Microbial colonization of the intestine in newborns is a critical event that regulates host physiology with lifelong consequences. However, there is a need to identify microbial molecules and mechanisms that play a role in this process. We aimed to elucidate the effects of flagellin, an important immunomodulatory component involved in bacterial motility, on intestinal epithelial functions. Methods: Germfree mice were colonized with synthetic bacterial communities, including a flagellated mouse gut isolate of Escherichia coli or a ΔfliC mutant thereof that lacks flagella. The offspring of these mice were studied at 7 days of age by single-cell RNA sequencing of epithelial cells isolated from the small intestine. Results: Enterococcus faecalis was the most dominant species in the small intestine of newborn mice. Blautia pseudococcoides was detected at low relative abundances. Colonization by either E. coli strain reduced the relative abundance of E. faecalis to 10% on average, and E. coli became the dominant member, with no substantial differences between mice colonized with the wildtype or mutant strain. The distribution of cell types within the small intestinal epithelium was skewed toward specialized cells (goblet, enteroendocrine, stem cells) at the expense of enterocytes in mice colonized with flagellated bacteria. These mice were characterized by higher expression of genes involved in cell-cell interactions (Npl, Epcam) and responses to infection (Ctsl) in enterocytes and enhanced translation (ribosomal proteins) in stem cells. In contrast, genes involved in lipid homeostasis (transport and metabolism; Apoc 2/4, Dgat2, Fabp 1/2, Mttp) in all cell types and inflammatory and androgen-related pathways in specialized cell types were less expressed in epithelial cells exposed to wildtype E. coli. Conclusion: These findings suggest that early life exposure to flagellated commensal bacteria influences cellular processes underlying tissue metabolism and remodeling in the small intestine. To investigate the effect of flagellin on intestinal epithelial functions, germfree mice were colonized with OMM9, OMM9+flagellated E. coli and OMM9+ E. coli mutant thereof that lacks flagella. The offspring of these mice were studied at 7 days of age by single-cell RNA sequencing of epithelial cells isolated from the small intestine.