Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence

Abstract

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

If looks could kill: Fungal macroscopic morphology and virulence

“…Examples of bacterial pathogens include V. cholerae [25], Burkholderia pseudomallei [5], nontuberculosis Mycobacterium spp. [26,27], Clostridium difficile [28], P. aeruginosa [8,29], Staphylococcus aureus [30,31], and N. gonorrhoeae [32] (Fig 2). Distinct bacterial and fungal colony morphotypes are also observed in clinical samples from specific body sites.…”

The impact of pH onClostridioides difficilesporulation and physiology

“…Differences in phenotypes for cells grown on solid and liquid medium have been observed for B. cereus , with plate-grown cells displaying increased gamma radiation resistance and a more developed S-layer compared to cells grown in liquid (70). Other studies of C. difficile found substantial differences in gene expression profiles between biofilms grown in broth and grown on plates (71), as well as differences in the expression of phase variation genes and the orientation of invertible elements (42, 72). We anticipate that differences in gene expression for liquid and solid medium affected the survival and adaptation of C. difficile after an acid shock; however, this was not explored in this study.…”

Enhancing bacterial survival through phenotypic heterogeneity

“…Inversion of the elements occurs stochastically and independently, yielding multiple discrete genetic combinations and phenotypic variants, thus allowing one recombinase to influence bacterial phenotypes on a broader scale. As an example, in C. difficile, the recombinase RecV inverts sequences upstream of multiple genes including those encoding CwpV, flagella, and intracellular factors [19,22,31]. Bottom: Phase variation of a single regulator, such as a transcription factor, results in a coordinated switch in multiple phenotypes regulated by that factor.…”

“…The bvgS gene contains a cytosine tract in its coding region and SSR-mediated variation in the number of cytosines affects BvgS production and transcription of BvgAS-regulated virulence genes [17,18]. In C. difficile, two invertible elements lie upstream of the colony morphology regulator genes cmrRST and the flagellar flgB operon, which each encode transcriptional regulators [8,19]. The cmrRST operon encodes a noncanonical TCS composed of a predicted histidine kinase, CmrS, and two putative response regulators with DNA binding domains, CmrR and CmrT [19].…”

“…In C. difficile, two invertible elements lie upstream of the colony morphology regulator genes cmrRST and the flagellar flgB operon, which each encode transcriptional regulators [8,19]. The cmrRST operon encodes a noncanonical TCS composed of a predicted histidine kinase, CmrS, and two putative response regulators with DNA binding domains, CmrR and CmrT [19]. CmrRST modulates multiple C. difficile phenotypes: colony morphology, biofilm formation, swimming motility, cell shape, and virulence in a model of acute disease [19].…”