Phase variation of Clostridium difficile virulence factors

Clostridium difficile is a leading cause of nosocomial infections, causing disease that ranges from mild diarrhea to potentially fatal colitis. A variety of surface proteins, including flagella, enable C. difficile colonization of the intestine. Once in the intestine, toxigenic C. difficile secretes two glucosylating toxins, TcdA and TcdB, which elicit inflammation and diarrheal disease symptoms. Regulation of colonization factors and TcdA and TcdB is an intense area of research in C. difficile biology. A recent publication from our group describes a novel regulatory mechanism that mediates the ON/OFF expression of co-regulated virulence factors of C. difficile, flagella and toxins. Herein, we review key findings from our work, present new data, and speculate the functional consequence of the ON/OFF expression of these virulence factors during host infection

Characterization of flagellum and toxin phase variation in Clostridioides difficile ribotype 012 isolates

Clostridioides difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum expression and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a flagellar switch upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting that the strain is phase locked. To determine whether a phase-locked flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase-off, nonmotile, and attenuated for toxin production. We isolated low-frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase-on derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains and perhaps other ribotypes, which could impact disease progression and diagnosis

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

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