Clostridioides difficile is Chemoattracted to Oligosaccharides Released by Mucin‐ Degrading Microbes

Background Clostridioides difficile is an enteric pathogen causing post‐antibiotic diarrhea and colitis. It is widely accepted that C. difficile exploits an intestinal environment with an altered microbiota, but the details of these microbe‐microbe interactions are not well characterized. Several pathogens have been demonstrated to adhere and colonize the intestinal mucus layer. However, few studies have addressed this interplay with C. difficile . We hypothesized that C. difficile would adhere to the human mucin MUC2 and that mucin‐associated microbes, which can liberate mucin glycan oligosaccharides, would promote C. difficile growth. Methods & Results Using CFDA‐SE fluorescently tagged bacteria, we demonstrated in vitro that C. difficile is capable of adhering to human MUC2 glycans. In vivo we identified co‐localization of C. difficile with colonic MUC2 in infected mice and surgical resections from C. difficile infected patients. To identify microbes that may be interacting with C. difficile we generated bioreactor communities with human stool with the addition of mucin‐coated inserts. 16S rRNA sequencing identified that C. difficile formed biofilms with several mucin‐degrading microbes. We reasoned that these microbes may be releasing mucin monosaccharides that might serve as a chemoattractant. C. difficile chemotaxed towards human MUC2 as well as mucin monosaccharides fucose, mannose, galactose, GalNAc, GlcNAc and sialic acid. The highest chemotaxis was observed with mannose and GlcNAc. We confirmed our chemotaxis phenotype using live cell imaging. To address whether C. difficile was capable of cleaving mucin glycans, we queried 35 C. difficile genomes and found that all C. difficile strains lacked the specific glycosyl hydrolases required to enzymatically cleave mucin O‐linked glycans. In contrast, the genomes of mucin degrading microbes identified from our 16S sequencing harbored an extensive repertoire of mucin‐degrading glycosyl hydrolases. In vitro , we confirmed that C. difficile was unable to degrade mucin glycans and use them as a primary carbon source for growth. However, co‐cultures with the mucin‐degrading Akkermansia muciniphila, Ruminococcus torques and Bacteroides thetaioatomicron promoted C. difficile growth in media containing purified MUC2, but lacking glucose. qPCR analysis of chemotaxis and adherence genes revealed an upregulation of key genes in response to both MUC2 and microbe‐mucin metabolites. Consistent with changes in chemotaxis genes, we observed that C. difficile chemotaxed toward mucin oligosaccharides released by A. muciniphila and B. thetaioatomicron.Conclusions These studies suggest that C. difficile is chemoattracted to and adheres to mucins, where it interacts with mucin‐degrading microbes which likely promote its colonization.