Phase variation of a signal transduction system controlsClostridioides difficilecolony morphology, motility, and virulence

Abstract

23di-GMP, biofilm 2 24 Abstract 25Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal 26 disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase 27 variation. Many C. difficile strains representing multiple ribotypes develop two colony 28 morphotypes, termed rough and smooth, but the biological implications of this phenomenon 29 have not been explored. Here, we examine the molecular basis and physiological relevance 30 of the distinct colony morphotypes produced by this bacterium. We show that C. difficile 31 reversibly differentiates into rough and smooth colony morphologies, and that bacteria 32 derived from the isolates display opposing surface and swimming motility behaviors. We 33identified an atypical phase-variable signal transduction system consisting of a histidine 34 kinase and two response regulators, named herein CmrRST, which mediates the switch in 35 colony morphology and motility behaviors. The CmrRST-regulated surface motility is 36 independent of Type IV pili, suggesting a novel mechanism of surface expansion in C. 37difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes 38 the formation of elongated bacteria arranged in bundled chains, which may contribute to 39 bacterial migration. In a hamster model of acute C. difficile disease, colony morphology 40 correlates with virulence, and the CmrRST system is required for disease development. 41Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting 42 that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. 43Our findings indicate that C. difficile employs phase variation of the CmrRST signal 44 transduction system to generate phenotypic heterogeneity during infection, with 45 concomitant effects on bacterial physiology and pathogenesis. 46 3 47 Significance Statement 48 Phenotypic heterogeneity within a genetically clonal population allows many mucosal 49 pathogens to survive within their hosts, balancing the need to produce factors that 50 promote colonization and persistence with the need to avoid the recognition of those 51 factors by the host immune system. Recent work suggests that the human intestinal 52 pathogen Clostridium difficile employs phase variation during infection to generate a 53 heterogeneous population differing in swimming motility, toxin production, and more. 54 This study identifies a signal transduction system that broadly impacts C. difficile 55 physiology and behavior in vitro and in an animal model. Phase variation of this system 56 is therefore poised to modulate the coordinated expression of multiple mechanisms 57 influencing C. difficile disease development. 4 58 59Phenotypic heterogeneity within bacterial populations is a widely established 60 phenomenon that allows the population to survive sudden environmental changes (1-3). 61Heterogeneity serves as a "bet-hedging" strategy such that a subpopulation can persist 62 and propagate an advantageo...

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

“…Another c-di-GMP-responsive riboswitch, Cdi2_2, precedes the genes encoding the signal transduction system CmrRST in C. difficile , where CmrS is a putative histidine kinase, and CmrR and CmrT are two predicted response regulators [ 181 ]. Binding of c-di-GMP to Cdi2_2 positively affects cmrRST expression [ 182 ], most likely through anti-termination of transcription. Interestingly, cmrRST is also regulated by phase variation with an invertible DNA sequence, the cmr switch, located upstream of the corresponding genes [ 182 ].…”

“…Binding of c-di-GMP to Cdi2_2 positively affects cmrRST expression [ 182 ], most likely through anti-termination of transcription. Interestingly, cmrRST is also regulated by phase variation with an invertible DNA sequence, the cmr switch, located upstream of the corresponding genes [ 182 ]. CmrRST modulates colony morphology (rough or smooth) with the activation of cmrRST expression by c-di-GMP binding to Cdi2_2, promoting the development of rough colonies.…”

“…CmrRST also inversely regulates motility and surface migration and promotes bacterial chaining. Importantly, a cmrR deletion mutant is unable to colonize the intestinal tract and has a strong virulence defect in the hamster model of infection [ 182 ].…”

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

“…difficile NAP1/B1/027 ribotype strain R20291, and an eighth in several 017 ribotype strains [ 43 , 44 ]. The identified sequences were shown to undergo inversion, and the sequence upstream of the cmrRST operon was confirmed to regulate expression of the downstream genes in a manner consistent with phase variation [ 44 , 45 ]. We subsequently showed that site-specific recombination also mediates phase variation of flagella and toxins by inversion of a genetic sequence called the “flagellar switch” [ 33 ].…”

“…difficile R20291 in broth culture consists of a heterogeneous population of flg ON and OFF bacteria. Notably, RecV is also required for inversion of the cwpV switch, as well as two of the other identified invertible sequences including one shown to impact multiple phenotypes including virulence [ 40 , 44 , 45 ].…”