SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Putnam, Emily E.; Nock, Adam M.; Lawley, Trevor D.; Shen, Aimee

doi:10.1128/jb.02181-12

Cited by 141 publications

(345 citation statements)

References 50 publications

(95 reference statements)

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“…In a sigH mutant, transcription of the opp genes was decreased, while app transcripts were increased, suggesting that the transporters are regulated differently during the adaptation to stationary phase in TY medium (60). However, our transcriptional analysis of app and opp expression in cells grown in sporulation medium (70:30 broth) (36) revealed that the expression of appA and oppB genes increases similarly throughout the transition to stationary phase (see Fig. S2A in the supplemental material).…”

Section: Identification Of Opp and App Transporters In C Difficilementioning

confidence: 72%

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Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014

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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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Section: Identification Of Opp and App Transporters In C Difficilementioning

confidence: 72%

“…To prepare spores for animal studies, strains for spore preparation were grown in BHIS broth overnight and 100 l was spread onto 70:30 agar plates (36). The plates were then incubated for 48 h to allow spores to form.…”

mentioning

confidence: 99%

Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014

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“…Whole-genome SNP analysis by Illumina next-generation sequencing revealed no additional nucleotide changes in the CD1492 mutant (see Materials and Methods). The CD1492 mutant was tested for the ability to sporulate on 70:30 sporulation agar (27). As demonstrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…C. difficile cultures were started in BHIS medium supplemented with 0.1% taurocholate to allow for germination of spores within the starting inoculum and 0.2% fructose to prevent sporulation. When cultures reached an optical density at 600 nm (OD 600 ) of 0.5, 250 l was applied evenly to 70:30 agar medium and incubated anaerobically at 37°C (27). Samples were scraped from the plates at the time points indicated for each experiment and evaluated for sporulation frequency by both phase-contrast counting and enumeration of ethanol-resistant spores.…”

Section: Methodsmentioning

confidence: 99%

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

et al. 2016

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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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“…C. difficile strains were started as lowdensity cultures in BHIS medium supplemented with 0.1% taurocholate (Sigma-Aldrich) and 0.5% fructose and were grown to mid-log phase. Cultures were subsequently back-diluted to an optical density at 600 nm (OD 600 ) of 0.5 in 70:30 sporulation medium (50) or BHIS medium. The cultures were then immediately diluted 1:10 into the main culture flask containing 70:30 or BHIS broth to reach a starting OD 600 of 0.05.…”

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confidence: 99%

CodY-Dependent Regulation of Sporulation in Clostridium difficile

et al. 2016

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Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile. The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile. IMPORTANCEThis study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile. We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains.

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SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Cited by 141 publications

References 50 publications

Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

CodY-Dependent Regulation of Sporulation in Clostridium difficile

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