Unique regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens

Abstract

Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).

Fig 1

Effects of a virX mutation on transcription of sporulation-specific sigma factors of C. perfringens strains. (A) The levels of sigF, sigE/sigG, and sigK transcripts in total RNAs isolated from 4-h nonsporulating GAM cultures were analyzed by Northern blotting. Lanes: 1, strain 13(pJIR418) (wild type); 2, TS186(pJIR418) (virX_strain13 mutant); 3, TS186(pTS907) (TS186 complemented with wild-type virX_strain13). (B) Northern blot results are shown for the transcription of spo0A, sigF, sigE/sigG, sigK, and virX during early logarithmic growth (1.5 and 2.5 h) and logarithmic growth (3.5, 4.5, and 5.5 h) of DS sporulation cultures. Lanes: 1, SM101 (wild type); 2, KO101 (virX_SM101 mutant); 3, KO102 (KO101 complemented with wild-type virX_SM101).

Fig 2

Sporulation efficiencies of various C. perfringens strains. To measure sporulation efficiency, 6- and 24-h sporulating cultures of SM101 (wild type), KO101 (virX mutant), KO102 (complemented with wild-type virX), KO103 (complemented with virX-K4stop), and KO104 (complemented with virX-K42stop) were directly plated onto BHI agar to determine the total numbers of CFU. The cultures were then heat treated (70°C, 20 min) and plated onto BHI agar to determine the numbers of CFU from heat-resistant spores. Both were incubated overnight under anaerobic conditions at 37°C. Sporulation efficiency (%) was calculated as follows: (heat-resistant CFU ml⁻¹/total CFU ml⁻¹) × 100. The data are presented as means ± standard deviations (SD) calculated from at least 3 independent experiments.

Fig 3

Cell morphologies of C. perfringens SM101 and its derivatives grown under sporulation conditions. Morphologies of 6-h DS sporulation cultures of C. perfringens SM101 (wild type), KO101 (virX mutant), and KO102 (KO101 complemented with wild-type virX) were analyzed by phase-contrast microscopy and fluorescence microscopy with FM4-64.

Fig 4

Effects of virX mutation on transcription and production of C. perfringens enterotoxin. (A) Total RNA was extracted at various times from DS sporulation cultures and analyzed by Northern blotting. Lanes: 1, SM101 (wild type); 2, KO101 (virX mutant); 3, KO102 (KO101 complemented with wild-type virX). (B) Eight-hour DS sporulation cultures of C. perfringens strains were analyzed by Western blotting using rabbit CPE antiserum. Lanes: 1, SM101 (wild type); 2, KO101 (virX mutant); 3, KO102 (KO101 complemented with wild-type virX).