c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

• Type of Material: Article
• Authors: Adrianne Edwards, Emory University; Caitlin L Willams, University of North Carolina; Nivedita Pareek, University of North Carolina; Shonna McBride, Emory University; Rita Tamayo, University of North Carolina
• Language: English
• Date: 2021-11-01
• Publisher: AMER SOC MICROBIOLOGY
• Publication Version: Final Published Version
• Copyright Statement: © 2021 Edwards et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1128/mSphere.00919-21
• Title of Journal or Parent Work: MSPHERE
• Volume: 6
• Issue: 6
• Start Page: e0091921
• End Page: e0091921
• Grant/Funding Information: This research was supported by the U.S. National Institutes of Health through research grants AI107029 and AI143638 to R.T. and AI116933 and AI156052 to S.M.M. C.L.W. is supported by an Institutional Research and Academic Career Development Award (IRACDA) fellowship under K12-GM000678. The content of the manuscript is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health.
• Keywords: VIRULENCE; anaerobe; REGULATOR; spore; CONSTRUCTION; Life Sciences & Biomedicine; INITIATION; Clostridioides difficile; cyclic diguanylate; c-di-GMP; IDENTIFICATION; cyclic diguanylate synthase; INTEGRATION; BACILLUS-SUBTILIS; Clostridium difficile; Microbiology; sporulation; TRANSCRIPTION; GENE; Science & Technology; AGGREGATION
• Research Categories: Health Sciences, Immunology; Biology, Microbiology

Abstract

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One second-messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. The overexpression of a gene encoding a diguanylate cyclase, dccA, decreased the sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630Derm strains. The expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630Derm, suggesting that DccA contributes to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, the overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. c-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCE Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen’s life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic strain R20291 and the historical strain 630Derm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.