Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>

ABSTRACT 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-GM...

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Autores principales:	Robert W. McKee, Carissa K. Harvest, Rita Tamayo
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Clostridium difficile biofilms c-di-GMP cyclic diguanylate flagellar motility riboswitch Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0fc2ac90b3e846659cdc33732b9a961d
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spelling	oai:doaj.org-article:0fc2ac90b3e846659cdc33732b9a961d2021-11-15T15:22:26ZCyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>10.1128/mSphere.00423-182379-5042https://doaj.org/article/0fc2ac90b3e846659cdc33732b9a961d2018-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00423-18https://doaj.org/toc/2379-5042ABSTRACT 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. difficile. IMPORTANCE 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.Robert W. McKeeCarissa K. HarvestRita TamayoAmerican Society for MicrobiologyarticleClostridium difficilebiofilmsc-di-GMPcyclic diguanylateflagellar motilityriboswitchMicrobiologyQR1-502ENmSphere, Vol 3, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridium difficile biofilms c-di-GMP cyclic diguanylate flagellar motility riboswitch Microbiology QR1-502
spellingShingle	Clostridium difficile biofilms c-di-GMP cyclic diguanylate flagellar motility riboswitch Microbiology QR1-502 Robert W. McKee Carissa K. Harvest Rita Tamayo Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
description	ABSTRACT 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. difficile. IMPORTANCE 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.
format	article
author	Robert W. McKee Carissa K. Harvest Rita Tamayo
author_facet	Robert W. McKee Carissa K. Harvest Rita Tamayo
author_sort	Robert W. McKee
title	Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
title_short	Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
title_full	Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
title_fullStr	Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
title_full_unstemmed	Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>
title_sort	cyclic diguanylate regulates virulence factor genes via multiple riboswitches in <italic toggle="yes">clostridium difficile</italic>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/0fc2ac90b3e846659cdc33732b9a961d
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Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>

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