AbstractRecent work has revealed thatClostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. ManyC. difficilestrains 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 thatC. difficilereversibly differentiates into rough and smooth colony morphologies, and that bacteria derived from the isolates display opposing surface and swimming motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein CmrRST, which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of Type IV pili, suggesting a novel mechanism of surface expansion inC. 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. In a hamster model of acuteC. difficiledisease, colony morphology correlates with virulence, and 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-expressingC. difficile. Our findings indicate thatC. difficileemploys phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.Significance StatementPhenotypic heterogeneity within a genetically clonal population allows many mucosal pathogens to survive within their hosts, balancing the need to produce factors that promote colonization and persistence with the need to avoid the recognition of those factors by the host immune system. Recent work suggests that the human intestinal pathogenClostridium difficileemploys phase variation during infection to generate a heterogeneous population differing in swimming motility, toxin production, and more. This study identifies a signal transduction system that broadly impactsC. difficilephysiology and behaviorin vitroand in an animal model. Phase variation of this system is therefore poised to modulate the coordinated expression of multiple mechanisms influencingC. difficiledisease development.
Diversity and Sociality Formed by Phenotypic Heterogeneity in a Clonal Population: Origins and Significance of Non-Genetic Individuality in Microbes
