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C-di-GMP Inhibits Early Sporulation in Clostridioides difficile.

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 630Δerm 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 630Δerm, 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 630Δerm. 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.

SUBMITTER: Edwards AN

PROVIDER: S-EPMC8653836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The ability of the anaerobic gastrointestinal pathogen Clostridioides difficile to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a predicted histidine kinase, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional predicted orphan histidine kinases CD2492, CD1579, and CD1949, which are hypothesized to regulate Spo0A phosphorylation. Disruption of CD2492 also increased sporulation frequency, similarly to the CD1492 mutant and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of CD1579 variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492, and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant-negative hypersporulation phenotype. Altogether, at least three predicted histidine kinases, CD1492, CD2492, and CD1579 (herein, PtpA, PtpB and PtpC), repress C. difficile sporulation initiation by regulating activity of Spo0A. IMPORTANCE The formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two predicted histidine kinase proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another predicted histidine kinase protein, CD1579 (PtpC), also represses sporulation and present evidence that a fourth predicted histidine kinase protein, CD1949, does not impact sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.

Project description:The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation.IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Project description:Clostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data, we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δerm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630Δerm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen.IMPORTANCEClostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630Δerm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce C. difficile growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.

Project description:The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

Project description:Short-chain fatty acids (SCFAs) are products of bacterial fermentation that help maintain important gut functions such as maintenance of the intestinal barrier, cell signaling, and immune homeostasis. The main SCFAs acetate, propionate, and butyrate have demonstrated beneficial effects for the host, including its importance in alleviating infections caused by pathogens such as Clostridioides difficile. Despite the potential role of SCFAs in mitigating C. difficile infection, their direct effect on C. difficile remains unclear. Through a set of in vitro experiments, we investigated how SCFAs influence C. difficile growth, sporulation, and toxin production. Similar to previous studies, we observed that butyrate decreased growth of C. difficile strain 630 in a dose-dependent manner. The presence of butyrate also increased C. difficile sporulation, with minimal increases in toxin production. RNA-Seq analysis validated our experimental results, demonstrating increased expression of sporulation-related genes in conjunction with changes in metabolic and regulatory genes, such as a putative carbon starvation protein, CstA. Collectively, these data suggest that butyrate may induce alternative C. difficile survival pathways, modifying its growth ability and virulence to persist in the gut environment. IMPORTANCE Several studies suggest that butyrate may modulate gut infections, such as reducing inflammation caused by the healthcare-associated Clostridioides difficile. While studies in both animal models and human studies correlate high levels of butyrate with reduced C. difficile burden, the direct impact of butyrate on C. difficile remains unclear. Our study demonstrates that butyrate directly influences C. difficile by increasing its sporulation and modifying its metabolism, potentially using butyrate as a biomarker to shift survival strategies in a changing gut environment. These data point to additional therapeutic approaches to combat C. difficile in a butyrate-directed manner.

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C-di-GMP Inhibits Early Sporulation in Clostridioides difficile.

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