AUTHOR=Zhu Duolong , Wang Shaohui , Sun Xingmin 

TITLE=FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.735616

DOI=10.3389/fmicb.2021.735616

ISSN=1664-302X

ABSTRACT=<p><italic>Clostridioides difficile</italic> flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during <italic>in vivo</italic> infection. However, how <italic>fliC</italic> expression is regulated in <italic>C. difficile</italic> remains unclear. In <italic>Bacillus subtilis</italic>, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism “FliW-CsrA-Hag.” In this study, we characterized FliW and CsrA functions by deleting or overexpressing <italic>fliW</italic>, <italic>csrA</italic>, and <italic>fliW</italic>-<italic>csrA</italic> in <italic>C. difficile</italic> R20291. We showed that <italic>fliW</italic> deletion, <italic>csrA</italic> overexpression in R20291, and <italic>csrA</italic> complementation in R20291ΔWA (<italic>fliW</italic>-<italic>csrA</italic> codeletion mutant) dramatically decreased FliC production, but not <italic>fliC</italic> gene transcription. Suppression of <italic>fliC</italic> translation by <italic>csrA</italic> overexpression can be relieved mostly when <italic>fliW</italic> was coexpressed, and no significant difference in FliC production was detected when only <italic>fliW</italic> was complemented in R20291ΔWA. Further, loss of <italic>fliW</italic> led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of <italic>C. difficile</italic> infection (CDI), while <italic>fliW</italic>-<italic>csrA</italic> codeletion decreased toxin production and mortality <italic>in vivo</italic>. Our data suggest that CsrA negatively modulates <italic>fliC</italic> expression and FliW indirectly affects <italic>fliC</italic> expression through inhibition of CsrA post-transcriptional regulation. In light of “FliW-CsrA-Hag” switch coregulation mechanism reported in <italic>B. subtilis</italic>, our data also suggest that “FliW-CsrA-<italic>fliC</italic>/FliC” can regulate many facets of <italic>C. difficile</italic> R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in <italic>C. difficile</italic>.</p>