FliW and CsrA govern flagellin (FliC) synthesis and play pleiotropic roles in virulence and physiology of Clostridioides difficile R20291

Abstract

Clostridioides difficile is a Gram-positive, spore-forming, and toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. In C. difficile, the expression of flagellar genes is coupled to toxin gene regulation and bacterial colonization and virulence. The flagellin FliC is responsible for pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated is unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor FliW, Flagellin Hag (FliC homolog), and CsrA (Carbon storage regulator A), which is referred to as partner-switching mechanism "FliW-CsrA-Hag". In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that both fliW deletion or csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion) dramatically decreased FliC production, however, fliC gene transcription was unaffected. While suppression of fliC translation by csrA overexpression was mostly relieved when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Taken together, these data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation, which seems similar to the "FliW-CsrA-Hag" switch in B. subtilis. Our data also suggest that "FliW-CsrA-fliC/FliC" can regulate many facets of C. difficile R20291 pathogenicity.