CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile.
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ABSTRACT:
SUBMITTER: Martins D
PROVIDER: S-EPMC8041843 | biostudies-literature | 2021 Apr
REPOSITORIES: biostudies-literature
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CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile.
Martins Diogo D DiCandia Michael A MA Mendes Aristides L AL Wetzel Daniela D McBride Shonna M SM Henriques Adriano O AO Serrano Mónica M
Scientific reports 20210412 1
Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50-60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We repor ...[more]
Similar Datasets
Project description:The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile.
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Project description:Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies.
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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.
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Project description:The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species.
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Project description:Clostridioides difficile is a Gram-positive, anaerobic bacterium. It is known that C. difficile is one of the major causes of antibiotic associated diarrhea. The enhanced antibiotic resistance observed in C. difficile is the result of highly resistant spores produced by the bacterium. In Bacillus subtilis, the sin operon is involved in sporulation inhibition. Two proteins coded within this operon, SinR and SinI, have an antagonistic relationship; SinR acts as an inhibitor to sporulation whereas SinI represses the activity of SinR, thus allowing the bacterium to sporulate. In a previous study, we examined the sin locus in C. difficile and named the two genes associated with this operon sinR and sinR', analogous to sinR and sinI in B. subtilis, respectively. We have shown that SinR and SinR' have pleiotropic roles in pathogenesis pathways and interact antagonistically with each other. Unlike B. subtilis SinI, SinR' in C. difficile carries two domains: the HTH domain and the Multimerization Domain (MD). In this study, we first performed a GST Pull-down experiment to determine the domain within SinR' that interacts with SinR. Second, the effect of these two domains on three phenotypes; sporulation, motility, and toxin production was examined. The findings of this study confirmed the prediction that the Multimerization Domain (MD) of SinR' is responsible for the interaction between SinR and SinR'. It was also discovered that SinR' regulates sporulation, toxin production and motility primarily by inhibiting SinR activity through the Multimerization Domain (MD).
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Project description: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.
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| S-EPMC7549369 | biostudies-literature
Project description:Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
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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.
| S-EPMC6997743 | biostudies-literature
Project description:Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101-1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin-receptor interaction.
| S-EPMC6212469 | biostudies-literature