Control of Clostridioides difficile virulence and physiology by the flagellin homeostasis checkpoint FliC-FliW-CsrA in the absence of motility

Mutations affecting Clostridioides difficile flagellin (FliC) have been shown to be hypervirulent in animal models and display increased toxin production and alterations in central metabolism. The regulation of flagellin levels in bacteria is governed by a tripartite regulatory network involving fliC, fliW, and csrA, which creates a feedback system to regulate flagella production. Through genomic analysis of C. difficile clade 5 strains (non-motile), we identified they have jettisoned many of the genes required for flagellum biosynthesis yet retain the major flagellin gene fliC and regulatory gene fliW. We therefore investigated the roles of fliC, fliW, and csrA in the clade 5 ribotype 078 strain C. difficile 1015, which lacks flagella and is non-motile. Analysis of mutations in fliC, fliW, and csrA (and all combinations) on C. difficile pathogenesis indicated that FliW plays a central role in C. difficile virulence as animals infected with strains carrying a deletion of fliW showed decreased survival and increased disease severity. These in vivo findings were supported by in vitro studies showing that mutations impacting the activity of FliW showed increased toxin production. We further identified that FliW can interact with the toxin-positive regulator TcdR, indicating that modulation of toxin production via FliW occurs by sequestering TcdR from activating toxin transcription. Furthermore, disruption of the fliC-fliW-csrA network results in significant changes in carbon source utilization and sporulation. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in C. difficile pathogenesis and physiology independent of their functions in motility.