A Novel PilR/PilS Two-Component System Regulates Necrotic Enteritis Pilus Production in Clostridium perfringens

Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic pilR, agrB, and virR null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1pilR mutant, while production in its complement (CP1pilR+) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (P ≤ 0.05), whereas this activity was restored in the complemented strain (P > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (P > 0.05), whereas the complemented strains exhibited significantly reduced binding (P ≤ 0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon. IMPORTANCE Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.