Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile

Abstract

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained singlenucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity

“…Renewed interest in RDTT beyond the E. coli paradigm has spurred from the recent findings that rho inactivation also triggers genome-wide pervasive (mostly antisense) transcription in Gram-positive species such as Bacillus subtilis 34 , Staphylococcus aureus 8 , or Mycobacterium tuberculosis 9 . Contrary to early expectations, depletion of Rho in these species can have significant consequences, from changes in cell differentiation or virulence programs in Firmicutes [34][35][36] to rapid death for M. tuberculosis 9 . The recent discovery of conditional regulatory mechanisms involving RDTT in the industrially important bacterium Corynebacterium glutamicum 37 also pinpoints to RDTT regulation being more widespread and decisive than previously envisioned.…”

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium

“…Another important feature of posttranscriptional control in C. difficile are cis-regulatory RNA elements. Pioneering work has deciphered the function of genetic switches in the 5′ untranslated region (UTR) of the flgB operon, containing the early-stage flagellar genes, and the cell wall protein-encoding gene cwpV (29)(30)(31). Moreover, cyclic di-GMP-responsive riboswitches were shown to regulate biofilm formation and toxin production (9,(32)(33)(34).…”

Wolf in Sheep’s Clothing: Clostridioides difficile Biofilm as a Reservoir for Recurrent Infections

“…Among the other regulation mechanisms that might also be involved in controlling C. difficile biofilm formation, epigenetics may influence biofilm formation, as DNA methylation by the methyltransferase CamA repressed biofilm formation [ 233 ]. Phase variation mechanisms controlling flagellar motility, colony morphology, and phosphodiesterases (PDEs) involved in the homeostasis of c-di-GMP and the surface protein CwpV may also influence biofilm formation [ 234 , 235 , 236 , 237 , 238 ]. Indeed, creation of heterogeneity in c-di-GMP levels and surface proteins by phase variation could generate sub-populations within the biofilm, leading to division of labor or dispersion [ 239 ].…”