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 single-nucleotide 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.
Highlights
Clostridioides difficile is a gram-positive, spore-forming anaerobe and the leading cause of antibiotic-associated diarrheal disease [1]
Phenotypic heterogeneity maintained by phase variation allows bacterial subpopulations to overcome potentially detrimental stresses in the environment, contributing to bacterial survival
Phase variation of flagella and toxins in C. difficile suggests that maintaining heterogeneity of their production may be important for survival and virulence
Summary
Clostridioides difficile is a gram-positive, spore-forming anaerobe and the leading cause of antibiotic-associated diarrheal disease [1]. Disease is largely mediated by two large glucosylating exotoxins, TcdA and TcdB [3,4,5] These toxins inactivate members of the Rho family of GTPases, causing a loss of interaction between the GTPase and its downstream effectors including those controlling the integrity of the actin cytoskeleton [6, 7]. During infection of a host intestine, the toxins cause disruption of the epithelial barrier, leading to development of diarrheal symptoms, immune cell recruitment, and inflammation [10]. These toxins are necessary for development of diarrheal disease in animal models of infection [4, 11, 12]
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