The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603

Abstract

Clostridium perfringens type A strains producing enterotoxin (CPE) cause one of the most common bacterial food-borne illnesses, as well as many cases of non-food-borne human gastrointestinal disease. Recent studies have shown that an Agr-like quorum-sensing system controls production of chromosomally encoded alpha-toxin and perfringolysin O by C. perfringens, as well as sporulation by Clostridium botulinum and Clostridium sporogenes. The current study explored whether the Agr-like quorum-sensing system also regulates sporulation and production of two plasmid-encoded toxins (CPE and beta2 toxin) that may contribute to the pathogenesis of non-food-borne human gastrointestinal disease strain F5603. An isogenic agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, providing the first evidence that, in C. perfringens, this system can control production of plasmid-encoded toxins as well as chromosomally encoded toxins. This mutant also showed reduced production of alpha-toxin and perfringolysin O during vegetative growth. Importantly, when cultured in sporulation medium, the mutant failed to efficiently form spores and was blocked for CPE production. Complementation partially or fully reversed all phenotypic changes in the mutant, confirming that they were specifically due to inactivation of the agr locus. Western blots suggest that this loss of sporulation and sporulation-specific CPE production for the agrB null mutant involves, at least in part, Agr-mediated regulation of production of Spo0A and alternative sigma factors, which are essential for C. perfringens sporulation.

Fig. 1.

Intron-based mutagenesis to create an isogenic F5603 agrB mutant and construction of a complementing strain. (A) PCR confirmation of the isogenic agrB null mutant and complementing strain. Using internal agrB primers, this PCR assay amplifies an ∼500-bp product from the wild-type agrB gene, but due to the presence of a 900-bp intron insertion, it amplifies an ∼1.5-kb PCR product from an intron-disrupted agrB gene. (B) Southern blot hybridization of an intron-specific probe to DNA from wild-type F5603 or the agrB null mutant. DNA from each strain was digested with EcoRI prior to electrophoresis on a 1% agarose gel prior to blotting and hybridization with an intron-specific probe. Size of DNA fragments, in kilobases (kb), is shown at the left. (C) RT-PCR analyses for agrB expression by F5603, the F5603::agrB mutant, and the complementing F5603agrBcomp strain grown for 5 h in DS medium. First and last lanes show size markers. Lanes labeled with a plus sign (+) were from samples receiving reverse transcriptase, while lanes labeled with a minus sign (−) lacked reverse transcriptase to show the absence of DNA contamination.

Fig. 2.

Production of CPA and PFO by vegetative cells of wild-type F5603, the agrB null mutant, and the complementing strain growing on agar plates. The phospholipase C activity of CPA was detected in colonies growing on egg yolk agar plates (A), and the beta-hemolytic activity of PFO was detected on blood agar plates (B). Colonies of wild-type F5603 and the complementing strain showed a characteristic CPA-induced halo zone on egg yolk agar plates and PFO-induced β-hemolysis surrounding colonies grown on blood agar plates. However, the mutant failed to produce these zones.

Fig. 3.

Western blot analysis of toxin production by wild-type F5603, the isogenic agrB null mutant, and the complementing strain growing in TGY vegetative culture broth (A) or DS sporulation medium (B). Each strain was grown for 16 h in TGY broth or DS medium, as indicated. Panel A shows production of PFO, CPA (alpha-toxin), CPE, and CPB2 toxin expression in TGY broth. Panel B shows production of PFO, CPA, CPE, and CPB2 in DS sporulation medium. The molecular weight of each band is indicated on the left.

Fig. 4.

RT-PCR analyses for toxin gene expression by F5603, the F5603::agrB mutant, and the complementing strain growing in DS medium. RNA was isolated from cultures grown for 5 h in DS medium. (A) Expression of the pfoA gene encoding PFO; (B) expression of the cpa gene encoding alpha-toxin; (C) expression of the cpe gene encoding enterotoxin; (D) expression of the cpb2 gene encoding beta2 toxin. The first lanes in panels A and C and last lanes in panels B and D show size markers. Lanes labeled with a plus sign (+) were from samples receiving reverse transcriptase, while lanes labeled with a minus sign (−) lacked reverse transcriptase to show the absence of DNA contamination.

Fig. 5.

Western blot analysis of DS cultures for Spo0A, SigF, and SigG production by wild-type F5603, the agrB null mutant, and the complementing strain. Cells from 5-h DS cultures were collected and lysed. Those lysates were then Western blotted for Spo0A expression (A), SigF expression (B), and SigG expression (C). The molecular weight of each band is indicated on the left.