Abstract
In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a “flagellar switch” that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins (“flg phase ON”). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion (“flg phase OFF”). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.
Highlights
Clostridium difficile, a Gram-positive, spore-forming obligate anaerobe, is the leading cause of nosocomial disease in the North America, Europe and Australia [1,2]
The C. difficile PCR ribotype 027 group is associated with greater odds of diarrheal disease severity, outcome, and death compared to many other PCR ribotypes [7]
We identified RecV, which controls phase variation of the cell wall protein CwpV, as the recombinase responsible for inversion at the flagellar switch in both directions. Together these findings indicate that flagellar motility and the production of toxins are subject to phase variation in C. difficile, identifying an additional level of regulation of these linked processes
Summary
Clostridium difficile, a Gram-positive, spore-forming obligate anaerobe, is the leading cause of nosocomial disease in the North America, Europe and Australia [1,2]. The Centers for Disease Control and Prevention list C. difficile infections (CDI) as an urgent threat related to the use of antibiotics [3]. Antibiotic use perturbs the gastrointestinal microbiota that normally protects against CDI [1,4]. The rates of recurrence and mortality associated with CDI have increased in part due to the emergence of epidemic-associated strains with enhanced sporulation rates and toxin production [1,5,6]. The C. difficile PCR ribotype 027 group is associated with greater odds of diarrheal disease severity, outcome, and death compared to many other PCR ribotypes [7]. An understanding of bacterial physiology and genetics in C. difficile 027 ribotypes could reveal unique therapeutic or diagnostic targets to ameliorate severe CDI
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