Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production

ABSTRACT The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP...

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Autores principales:	Ummey Khalecha Bintha Ahmed, Tyler M. Shadid, Jason L. Larabee, Jimmy D. Ballard
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Clostridioides difficile Agr TcdA Cas9 autoinducing peptide Clostridium difficile Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9b1dc68adb34480c81d1c5a73a3b3f81
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spelling	oai:doaj.org-article:9b1dc68adb34480c81d1c5a73a3b3f812021-11-15T15:55:43ZCombined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production10.1128/mBio.03190-202150-7511https://doaj.org/article/9b1dc68adb34480c81d1c5a73a3b3f812020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.03190-20https://doaj.org/toc/2150-7511ABSTRACT The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action. IMPORTANCE C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile. These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr’s mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.Ummey Khalecha Bintha AhmedTyler M. ShadidJason L. LarabeeJimmy D. BallardAmerican Society for MicrobiologyarticleClostridioides difficileAgrTcdACas9autoinducing peptideClostridium difficileMicrobiologyQR1-502ENmBio, Vol 11, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridioides difficile Agr TcdA Cas9 autoinducing peptide Clostridium difficile Microbiology QR1-502
spellingShingle	Clostridioides difficile Agr TcdA Cas9 autoinducing peptide Clostridium difficile Microbiology QR1-502 Ummey Khalecha Bintha Ahmed Tyler M. Shadid Jason L. Larabee Jimmy D. Ballard Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
description	ABSTRACT The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action. IMPORTANCE C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile. These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr’s mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.
format	article
author	Ummey Khalecha Bintha Ahmed Tyler M. Shadid Jason L. Larabee Jimmy D. Ballard
author_facet	Ummey Khalecha Bintha Ahmed Tyler M. Shadid Jason L. Larabee Jimmy D. Ballard
author_sort	Ummey Khalecha Bintha Ahmed
title	Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
title_short	Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
title_full	Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
title_fullStr	Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
title_full_unstemmed	Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production
title_sort	combined and distinct roles of agr proteins in <named-content content-type="genus-species">clostridioides difficile</named-content> 630 sporulation, motility, and toxin production
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/9b1dc68adb34480c81d1c5a73a3b3f81
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Combined and Distinct Roles of Agr Proteins in <named-content content-type="genus-species">Clostridioides difficile</named-content> 630 Sporulation, Motility, and Toxin Production

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