Culturing and Maintaining &lt;em&gt;Clostridium difficile&lt;/em&gt; in an Anaerobic Environment

Edwards, Adrianne N.; Suárez, Jose M.; McBride, Shonna M.

doi:10.3791/50787

Cited by 82 publications

(76 citation statements)

References 35 publications

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“…Fructose was added to overnight cultures at 0.5% as needed to prevent sporulation. C. difficile was cultured anaerobically in a vinyl chamber (Coy Laboratory Products) at 37°C with an atmosphere of 10% H 2 , 5% CO 2 , and 85% N 2 as previously described (41,42). Escherichia coli strains were cultured at 37°C in LB (43) or BHIS medium supplemented with 20 g chloramphenicol ml Ϫ1 and 100 g ampicillin ml Ϫ1 as needed to maintain plasmid selection.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

CodY-Dependent Regulation of Sporulation in Clostridium difficile

et al. 2016

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“…Cultures were supplemented with 20 g chloramphenicol ml Ϫ1 (Sigma-Aldrich) or 100 g ampicillin ml Ϫ1 (Cayman Chemical Company) as needed. C. difficile strains were grown in brain heart infusion medium supplemented with 2% yeast extract (BHIS; Becton, Dickinson, and Company) or on BHIS agar plates (24) at 37°C in an anaerobic chamber (Coy Laboratory Products) as previously described (25)(26)(27). BHIS medium was supplemented with 0.6 to 1.0 mg lysozyme ml Ϫ1 (Fisher Scientific), 150 to 200 g polymyxin B ml Ϫ1 (Sigma-Aldrich), 2 g thiamphenicol ml Ϫ1 (Sigma-Aldrich), or 0.5 g kanamycin ml Ϫ1 or 0.5 g nisin ml Ϫ1 (MP Biomedicals) as needed.…”

Section: Methodsmentioning

confidence: 99%

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

et al. 2016

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Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibioticassociated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Hostproduced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of D-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor V does not induce dlt expression in response to lysozyme, indicating that V is required for regulation of lysozyme-dependent D-alanylation of the cell wall. Using reporter gene fusions and 5= RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall D-alanylation in C. difficile is induced by lysozyme in a V -dependent manner and that this pathway impacts virulence in vivo.C lostridium difficile (Peptoclostridium difficile) causes nearly half a million infections in the United States each year, representing a significant public health threat (1). In order to cause infection, C. difficile must colonize the colon. As an important interface between the host and microbiota, the colon is an environment rich in host innate immune molecules and bacteriumderived antimicrobials made by the indigenous microbiota (2-6). These innate immune molecules and bacterially produced antimicrobials include a variety of cationic antimicrobial peptides (CAMPs), such as lysozyme, defensins, and bacteriocins (2,4,[7][8][9]. Understanding how C. difficile is able to resist killing in this antimicrobial-laden environment could better our understanding of the factors that contribute to the progression of C. difficile infections.A common mechanism of resistance to CAMPs in many bacteria is the alteration of the cell surface charge (10-12). One mechanism for increasing the surface charge is through the addition of D-alanine (D-Ala) to teichoic acids in the cell wall (10,12,13). The addition of D-Ala is mediated by four proteins, DltA, DltB, DltC, and DltD, encoded by the dlt operon (13). The Dlt pathway confers lysozyme resistance to Bacillus subtilis and Enterococcus faecalis (14,15). Previously, we demonstrated that the D-alanylation of the cell wall via ...

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“…C. difficile cultures were grown in an anaerobic chamber (Coy Laboratory Products) containing an atmosphere of 85% nitrogen, 10% hydrogen, and 5% CO 2 at 37°C as described previously (17). C. difficile strains were cultured in brain heart infusion (BHI) medium supplemented with 2% yeast extract (BHIS medium) as broth or 1.5% agar medium (18).…”

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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Culturing and Maintaining <em>Clostridium difficile</em> in an Anaerobic Environment

Cited by 82 publications

References 35 publications

CodY-Dependent Regulation of Sporulation in Clostridium difficile

CodY-Dependent Regulation of Sporulation in Clostridium difficile

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

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Culturing and Maintaining <em>Clostridium difficile</em> in an Anaerobic Environment

Cited by 82 publications

References 35 publications

CodY-Dependent Regulation of Sporulation in Clostridium difficile

CodY-Dependent Regulation of Sporulation in Clostridium difficile

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ V in Response to Lysozyme

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

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The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme