Piepenbrink, K. H. | bioRxiv Science

bioRxiv2025

Dissecting the Role of Flagellar Subunits in Clostridioides difficile Mucosal Colonization

Clostridioides difficile is a common cause of acute gastrointestinal (GI) inflammation in mammals, which can have detrimental effects on host health. C. difficile associated disease (CDAD) requires the secretion of high-molecular weight toxins after colonization of the GI tract. The molecular mechanisms of GI colonization by C. difficile, include potential interactions with host cells and the mucus layer formed from secreted mucin glycoproteins. C. difficile associates with the mucus layer in vivo and will associate with both epithelial cells and mucosal surfaces in vitro. Previously, we found a substantial defect in binding to mucosal surfaces for mutants of the major flagellar subunit, fliC, while mutation of the major subunit of type IV pili, pilA1, showed increased adhesion. To elucidate the mechanisms by which C. difficile interacts with ex vivo 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 C. difficile R20291. We discovered that the pilA1 mutant showed increased flagellation, while decreases in flagellation were found for fliC, fliD, and flg-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 flg-ON mutant showed increased flagellation without a significant increase in adhesion. We also found that the fliC mutant was defective in binding to mucus-secreting HT-29 MTX cells, but not HT-29 cells. These results imply that at least two molecular pathways contribute to C. difficile mucosal adhesion. In addition to their direct roles encoding T4P and flagellar subunits, pilA1 and fliC may contribute to regulating other factors relevant to mucosal adhesion.

microbiology↗

bioRxiv2023

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile

Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is anaerobic bacterium which colonizes the mammalian GI tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucus layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well-understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed that defects in adhesion by mutants deficient in flagella, but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus.

microbiology↗