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Global analysis of the sporulation pathway of Clostridium difficile.

ABSTRACT: The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors ?(F), ?(E), ?(G), and ?(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of ?(F), ?(E), ?(G), and ?(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were ?(F)-dependent, 169 were ?(E)-dependent, 34 were ?(G)-dependent, and 31 were ?(K)-dependent. In contrast with B. subtilis, C. difficile ?(E) was dispensable for ?(G) activation, ?(G) was dispensable for ?(K) activation, and ?(F) was required for post-translationally activating ?(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.

SUBMITTER: Fimlaid KA

PROVIDER: S-EPMC3738446 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Global analysis of the sporulation pathway of Clostridium difficile.

Fimlaid Kelly A KA Bond Jeffrey P JP Schutz Kristin C KC Putnam Emily E EE Leung Jacqueline M JM Lawley Trevor D TD Shen Aimee A

PLoS genetics 20130808 8

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Des ...[more]

PMID: 23950727

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Project description:UNLABELLED:Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE:Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.

Project description:UnlabelledClostridium 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. difficileImportanceThis 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.

Dataset Information

Global analysis of the sporulation pathway of Clostridium difficile.

Publications

Global analysis of the sporulation pathway of Clostridium difficile.

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