Abstract
ABSTRACTWe identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration. In the present work, we isolated representative CSCM strains using selective media based upon our initial 16S rRNA-based molecular identification (i.e., Acinetobacter, Delftia, and Stenotrophomonas). Their tropism for the crypt was confirmed, and their influence on epithelial regeneration was demonstrated in vivo by monocolonization of germfree mice. We also showed that lipopolysaccharide (LPS), through its endotoxin activity, was the dominant bacterial agonist controlling proliferation. The relevant molecular mechanisms were analyzed using colonic crypt-derived organoids exposed to bacterial sonicates or highly purified LPS as agonists. We identified a Toll-like receptor 4 (TLR4)-dependent program affecting crypts at different stages of epithelial differentiation. LPS played a dual role: it repressed cell proliferation through RIPK3-mediated necroptosis of stem cells and cells of the transit-amplifying compartment and concurrently enhanced cell differentiation, particularly the goblet cell lineage.
Highlights
We identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration
Bacteria isolated from proximal colonic tissues were identified by phenotypic characterization using the Biolog system, followed by Sanger sequencing of the 16S rRNA gene and two housekeeping genes
We focused the present work on four representative species: A. modestus, A. radioresistens, D. tsuruhatensis, and S. maltophilia
Summary
We identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration. We isolated representative CSCM strains using selective media based upon our initial 16S rRNA-based molecular identification (i.e., Acinetobacter, Delftia, and Stenotrophomonas) Their tropism for the crypt was confirmed, and their influence on epithelial regeneration was demonstrated in vivo by monocolonization of germfree mice. IMPORTANCE The LPS from crypt-specific core microbiota controls intestinal epithelium proliferation through necroptosis of stem cells and enhances cell differentiation, mainly the goblet cell lineage. We recently identified members of the Acinetobacter, Stenotrophomonas, and Delftia genera in the crypts of the murine cecum and proximal colon We named this bacterial assemblage the “crypt-specific core microbiota” (CSCM) [9]. On the basis of our recent results demonstrating a Nod2-mediated cytoprotective pathway of gut stem cells by bacterial muramyl-dipeptide [10], we decided to address the possible roles of CSCM members on crypt physiology and to characterize these strains and their possible functional impact
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