The VirSR Two-Component Signal Transduction System Regulates NetB Toxin Production in Clostridium perfringens

Abstract

Clostridium perfringens causes several diseases in domestic livestock, including necrotic enteritis in chickens, which is of concern to the poultry industry due to its health implications and associated economic cost. The novel pore-forming toxin NetB is a critical virulence factor in the pathogenesis of this disease. In this study, we have examined the regulation of NetB toxin production. In C. perfringens, the quorum sensing-dependent VirSR two-component signal transduction system regulates genes encoding several toxins and extracellular enzymes. Analysis of the sequence upstream of the netB gene revealed the presence of potential DNA binding sites, or VirR boxes, that are recognized by the VirR response regulator. In vitro binding experiments showed that purified VirR was able to recognize and bind to these netB-associated VirR boxes. Furthermore, using a reporter gene assay, the netB VirR boxes were shown to be functional. Mutation of the virR gene in two avian C. perfringens strains was shown to significantly reduce the production of the NetB toxin; culture supernatants derived from these strains were no longer cytotoxic to Leghorn male hepatoma cells. Complementation with the virRS operon restored the toxin phenotypes to wild type. The results also showed that the VirSR two-component system regulates the expression of netB at the level of transcription. We postulate that in the gastrointestinal tract of infected birds, NetB production is upregulated when the population of C. perfringens cells reaches a threshold level that leads to activation of the VirSR system.

The Clostridium perfringens Germinant Receptor Protein GerKC Is Located in the Spore Inner Membrane and Is Crucial for Spore Germination

“…PCR was performed on SM101 DNA to retarget the intron by using gerKC-specific primers CPP779, CPP780, and CPP781 (see Table S2 in the supplemental material) and the LtrBAsEBS2 universal primer (CGAAATTAGAAACTTGCG TTCAGTAAAC) provided with the Targetron gene knockout system (Sigma-Aldrich Corporation, St. Louis, MO). An ϳ350-bp BsrGIHindIII fragment retargeted to position 468/469 in the sense orientation of gerKC was cloned between the BsrGI and HindIII sites of the pJIR3566 vector (20), giving plasmid pDP276. Plasmid pDP276 was electroporated (21) into C. perfringens strain SM101, and erythromycin (Em)-resistant (Em r ) transformants were selected on brain heart infusion (BHI) agar plates supplemented with 30 g/ml Em.…”

Toxin Plasmids of Clostridium perfringens

“…More recently, VirS/ VirR was found to control NetB and CPB production by type A or C strains, respectively (151)(152)(153). Interestingly, close contact with enterocyte-like Caco-2 cells increases production of CPB by type C strain CN3685, and this effect requires VirS/VirR (153).…”

Unique Regulatory Mechanism of Sporulation and Enterotoxin Production in Clostridium perfringens

“…perfringens possesses great toxin versatility and is capable of producing up to 18 different toxins (3,26,27). The majority of these toxins are under the control of regulatory mechanisms exclusive to the vegetative growth phase (16,20,28). Recent evidence has demonstrated that alpha-and theta-toxin production is also dependent on an Agr-like quorum-sensing system that plays roles in toxin regulation under vegetative growth (29,30) and is also required for efficient sporulation and production of CPE (11).…”