A genetic switch controls the production of flagella and toxins in Clostridium difficile

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.

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

“…In this mechanism, a clonal population of cells becomes genetically heterogenous owing to the generation of (reversible) genetic variants that arise spontaneously. Interestingly, one of the promoters driving production of σ TcdR is recognized by σ D , and σ D expression is subject to phase variation (Anjuwon‐Foster and Tamayo, ). The gene for σ D is located in the flgB operon, which contains flagellar genes and is required for motility (El Meouche et al, ; McKee et al, ; Anjuwon‐Foster and Tamayo, ).…”

“…Interestingly, one of the promoters driving production of σ TcdR is recognized by σ D , and σ D expression is subject to phase variation (Anjuwon‐Foster and Tamayo, ). The gene for σ D is located in the flgB operon, which contains flagellar genes and is required for motility (El Meouche et al, ; McKee et al, ; Anjuwon‐Foster and Tamayo, ). Expression of the flgB operon is regulated by a 154 bp invertible element flanked by 21 bp inverted repeats located between the promoter and the first gene of the operon.…”

“…Expression of the flgB operon is regulated by a 154 bp invertible element flanked by 21 bp inverted repeats located between the promoter and the first gene of the operon. It was recently demonstrated that when the invertible element is in the ON orientation σ D is produced, leading to increased expression of tcdR , which in turn drives expression of the toxin genes tcdA and tcdB (Anjuwon‐Foster and Tamayo, ). Conversely, when the element flips to the OFF orientation, little or no σ D is produced and expression of toxin genes is attenuated (Anjuwon‐Foster and Tamayo, ).…”

“…To explore the role of phase variable production of σ D in generating a bimodal distribution of toxin gene expression, we first asked whether σ D is required for expression of our P tcdA ::rfp reporter. To this end, we introduced the reporter plasmid into an R20291 sigD::erm mutant and its isogenic wild‐type parent (Anjuwon‐Foster and Tamayo, ). Eliminating σ D reduced the fraction of red fluorescent (TcdA‐ON) cells from ~40% to ~5% of the population, consistent with previous reports that assayed toxin gene expression in a bulk population of cell (Fig.…”

Second‐order cooperation: Cooperative offspring as a living public good arising from second‐order selection on non‐cooperative individuals

“…; Moxon et al. , ; Anjuwon‐Foster and Tamayo ). Going one step further, Colizzi and Hogeweg () raise the question of the ability of a single sequence to encode an ecosystem, where second‐order selection would shape the mutational landscape such that different likely mutants could stably interact together.…”