Dissecting the role of flagellar subunits in <i>C. difficile</i> mucosal colonization

<i>Clostridioides difficile</i> is a common cause of acute gastrointestinal (GI) inflammation in mammals, which can have detrimental effects on host health. <i>C. difficile</i>-associated disease requires the secretion of high-molecular-weight toxins after colonization of the GI tract. The molecular mechanisms of GI colonization by <i>C. difficile</i> include potential interactions with host cells and the mucus layer formed from secreted mucin glycoproteins. <i>C. difficile</i> associates with the mucus layer <i>in vivo</i> and will associate with both epithelial cells and mucosal surfaces <i>in vitro</i>. Previously, we found a substantial defect in binding to mucosal surfaces for mutants of the major flagellar subunit, <i>fliC</i>, while mutation of the major subunit of type IV pili, <i>pilA1</i>, showed increased adhesion. To elucidate the mechanisms by which <i>C. difficile</i> interacts with <i>ex vivo</i> mucosal surfaces, we have measured swimming motility, mucosal adhesion, and levels of flagellation by transmission electron microscopy for mutants of flagellar and T4P genes in <i>C. difficile</i> R20291. We discovered that the <i>pilA1</i> mutant showed increased flagellation, while decreases in flagellation were found for <i>fliC, fliD,</i> and <i>flg</i>-Δ3 OFF (a phase-locked mutant with low transcription of the F3 flagellar operon), which were associated with both low swimming motility and low adhesion to mucosal surfaces. However, the reversed <i>flg</i>-Δ3 ON mutant showed increased flagellation without a significant increase in adhesion. We also found that the <i>fliC</i> mutant was defective in binding to mucus-secreting HT-29 MTX cells, and that decreased binding was not observed for other mutants with reduced flagellation. These results imply that at least two molecular pathways contribute to <i>C. difficile</i> mucosal adhesion. In addition to their direct roles encoding T4P and flagellar subunits, <i>pilA1</i> and <i>fliC</i> may contribute to regulatory networks governing other proteins relevant to mucosal adhesion.IMPORTANCEIn the context of previous work on <i>Clostridioides difficile</i> host adhesion by our groups and others, our results suggest that (i) at least two mechanisms exist for mucosal adhesion by <i>C. difficile</i>, potentially direct adhesion by flagella and another lectin-like adhesion regulated by flagellar components; (ii) mucosal binding can also contribute to <i>C. difficile</i> adhesion in 2D cell culture and could explain previous defects from <i>fliC</i> mutants; and (iii) levels of flagellation are largely insensitive to <i>fliC</i> transcription, implying that other factors limit flagellar production and that FliC levels may be regulated independently as part of regulatory networks within <i>C. difficile</i>.