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Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

ABSTRACT: The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.

SUBMITTER: Shimizu T

PROVIDER: S-EPMC135029 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

Shimizu Takeshi T Shima Kensuke K Yoshino Ken-ichi K Yonezawa Kazuyoshi K Shimizu Tohru T Hayashi Hideo H

Journal of bacteriology 20020501 10

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system ...[more]

PMID: 11976286

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Project description:BACKGROUND: Clostridium perfringens is a Gram-positive anaerobic bacterium causing severe diseases such as gas gangrene and pseudomembranosus colitis, that are generally due to the secretion of powerful extracellular toxins. The expression of toxin genes is mainly regulated by VirR, the response regulator of a two-component system. Up to now few targets only are known for this regulator and mainly in one strain (Strain 13). Due to the high genomic and phenotypic variability in toxin production by different strains, the development of effective strategies to counteract C. perfringens infections requires methodologies to reconstruct the VirR regulon from genome sequences. RESULTS: We implemented a two step computational strategy allowing to consider available information concerning VirR binding sites in a few species to scan all genomes of the same species, assuming the VirR targets are at least partially conserved across these strains. Results obtained are in agreement with previous works where experimental validation of the promoters have been performed and showed the presence of a core and an accessory regulon of VirR in C. perfringens strains with three target genes also located on plasmids. Moreover, the type E strain JGS1987 has the largest predicted regulon with as many as 10 VirR targets not found in the other genomes. CONCLUSIONS: In this work we exploited available experimental information concerning the targets of the VirR toxin regulator in one C. perfringens strain to obtain plausible predictions concerning target genes in genomes and plasmids of nearby strains. Our predictions are available for wet-lab researchers working on less characterized C. perfringens strains that can thus design focused experiments reducing the search space of their experiments and increasing the probability of characterizing positive targets with less efforts. Main result was that the VirR regulon is variable in different C. perfringens strains with 4 genes controlled in all but one strains and most genes controlled in one or two strains only.

Project description:Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the present study tested whether ETX expression by type D strain CN3718 also requires these regulatory systems. The agr operon was shown to be essential for signaling CN3718 to produce ETX in broth culture or to upregulate ETX production upon close contact with enterocyte-like Caco-2 cells, which may have pathogenic relevance since ETX is produced intestinally. However, ETX production remained at wild-type levels after inactivation of the VirS/VirR system in CN3718. These findings provide the first information regarding regulation of ETX production and suggest Agr-like QS toxin production regulation in C. perfringens does not always require the VirS/VirR system.

Project description:UnlabelledHeat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to ?-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase.ImportanceClostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria.

Dataset Information

Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

Publications

Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

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