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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2018-10-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00423-18 |
| _version_ | 1818836536037212160 |
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| author | Robert W. McKee Carissa K. Harvest Rita Tamayo |
| author_facet | Robert W. McKee Carissa K. Harvest Rita Tamayo |
| author_sort | Robert W. McKee |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-19T03:08:10Z |
| format | Article |
| id | doaj.art-0fc2ac90b3e846659cdc33732b9a961d |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-19T03:08:10Z |
| publishDate | 2018-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-0fc2ac90b3e846659cdc33732b9a961d2022-12-21T20:38:04ZengAmerican Society for MicrobiologymSphere2379-50422018-10-013510.1128/mSphere.00423-18Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic>Robert W. McKee0Carissa K. Harvest1Rita Tamayo2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, USADepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, USADepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mSphere.00423-18Clostridium difficilebiofilmsc-di-GMPcyclic diguanylateflagellar motilityriboswitch |
| spellingShingle | Robert W. McKee Carissa K. Harvest Rita Tamayo Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in <italic toggle="yes">Clostridium difficile</italic> mSphere Clostridium difficile biofilms c-di-GMP cyclic diguanylate flagellar motility riboswitch |
| title | 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_short | 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 |
| topic | Clostridium difficile biofilms c-di-GMP cyclic diguanylate flagellar motility riboswitch |
| url | https://journals.asm.org/doi/10.1128/mSphere.00423-18 |
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