Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in Clostridium difficile

mSphere. 2018 Oct 24;3(5):e00423-18. doi: 10.1128/mSphere.00423-18.


The intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that in Clostridium difficile (also called Clostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP in C. difficile, employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formation in vitro A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as "off" switches in response to c-di-GMP, while class II riboswitches acted as "on" switches. Transcriptional analyses of genes 3' of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network in C. difficile and emphasizes the role of the riboswitches in controlling known and putative virulence factors in C. difficileIMPORTANCE In Clostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP in C. difficile Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functional in vivo and displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression in C. difficile.

Keywords: Clostridium difficile; biofilms; c-di-GMP; cyclic diguanylate; flagellar motility; riboswitch.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / genetics
  • Clostridioides difficile / genetics*
  • Clostridioides difficile / pathogenicity
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / genetics
  • Gene Expression Regulation, Bacterial / genetics*
  • Riboswitch / physiology*
  • Virulence / genetics*
  • Virulence Factors / biosynthesis
  • Virulence Factors / genetics*


  • Bacterial Proteins
  • Riboswitch
  • Virulence Factors
  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP