The enteric pathogen Clostridium difficile chemotaxes towards mucin glycans during the early colonization phase of infection

Background Infection with the pathogen Clostridium difficile is a major cause of antibiotic‐induced diarrhea and colitis. In the United States, C. difficile infects millions of patients each year and results in healthcare associated costs of over 1 billion dollars. Despite the significant burden of C. difficile infection, the exact mechanisms of C. difficile host colonization remain unknown. It is well known that antibiotic use sets the stage for infection and that toxin production induces symptoms. However, how C. difficile identifies and colonizes its ecological niche are unclear. Similar to other pathogens, flagellum‐mediated motility and the ability to interact with the intestinal mucus layer are likely critical factors for C. difficile colonization. We hypothesized that C. difficile would recognize mucin glycans present on human MUC2 and would chemotax towards these oligosaccharides. We also speculated that MUC2 would influence C. difficile gene expression and promote colonization. Methods & Results To address these gaps in knowledge we fluorescently tagged C. difficile R20291 with CFDA‐SE and measured chemotaxis using a standard capillary assay. We observed that C. difficile was able to chemotax towards purified human MUC2 from a variety of sources, including human goblet cell lines HT29‐MTX, LS174T, T84 as well as human stool. We also found that C. difficile could recognize oligosaccharides present on MUC2 glycans, such as fucose, galactose, mannose, GalNac, GluNAc, and neurominic acid. The greatest accumulation of C. difficile was observed with mannose. To confirm these findings, we live‐imaged C. difficile chemotaxis in IBIDI chemotaxis slides and found that the presence of MUC2 or mannose increased the velocity and distance traveled by C. difficile compared to buffer medium alone. Examination of C. difficile genomes from several ribotypes revealed that C. difficile lacks the glycosyl hydrolases (GH) required for O‐linked mucin glycan degradation, although it did contain GH family 38, which cleaves mannose residues. To assess whether C. difficile could use mucin oligosaccharides as a primary carbon source, we grew C. difficile in a fully defined minimal medium (CDMM) in the absence of glucose supplemented with individual glycan oligosaccharides or MUC2. Consistent with our GH analysis, C. difficile was unable to grow on intact MUC2 alone. However, C. difficile was able to utilize all mucin oligosaccharides for growth. Further analysis of the C. difficile genome confirmed the transporters required for uptake of these sugars are present in all species. Finally, RNAseq demonstrated shifts in gene expression in C. difficile R20291 in response to human MUC2. Conclusions These data indicate that C. difficile recognizes and utilizes mucin‐derived oligosaccharides. We also demonstrate that C. difficile does not possess the enzymatic capacity to degrade these glycans on its own. Therefore, it must require the presence of other mucin‐degrading microbes for growth, pointing to the role of the microbial community in supporting C. difficile colonization.