Flagellin modulates regulation ofClostridiodes difficilepathogenesis in the absence of motility

Abstract

AbstractClostridioides difficileflagellin (FliC) is associated with toxin gene expression, bacterial colonization and virulence, and is also involved in pleiotropic gene regulation duringin vivoinfection. However, howfliCexpression is regulated and how FliC modulatesC. difficilepathogenicity remain unclear. In other bacterial species, FliC participates in a regulatory network with FliW and CsrA to regulate motility. Currently, studies investigating the role offliCinC. difficilephysiology were performed with motile strains. Despite its canonical role in motility, we foundfliCis highly conserved in non-motileC. difficilestrains that have jettisoned most flagellar assembly genes. We therefore investigated the roles offliCin pathogenesis and physiology in the non-motile clade 5 ribotype 078 strainC. difficile1015 (CD1015). We determined thatfliCwas expressed in CD1015 and the regulatory role offliCon toxin production is independent of functional flagella and motility. We showed protein-protein interactions between FliW-FliC and FliW-CsrA using a bacterial two-hybrid system and identified the required binding site for CsrA post-transcriptional regulation in the 5’ untranslated region of thefliCtranscript. Analysis of mutations infliC,fliWandcsrA(and all combinations) onC. difficilepathogenesis indicated that FliW plays a central role inC. difficilevirulence as animals infected with strains carrying a deletion offliWshowed decreased survival and increased disease severity. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in pathogenesis independent of their functions in motility.Author SummaryC. difficileis a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation ofC. difficletoxin production by FliC has been demonstratedin vitroandin vivoand has been proposed to link motility and virulence. Here we show that clinically important, non-motileC. difficilestrains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility in the pathogenesis and physiology ofC. difficile.