Regulation of pilus production by the PilR/PilS two-component and Agr-like quorum sensing systems in Clostridium perfringens

Abstract

Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry. Their pilus plays a role in adhesion and is important in pathogenesis. The NE pilus in these isolates consists of three pilins (CnaA, FimA, FimB) encoded by three genes that belong to the VR-10B allele of the VR-10 locus. There are two genes downstream of the pilin genes encoding a two-component regulatory system (PilR/PilS). However, the regulatory function of PilR/PilS remains to be determined. The current study has investigated the function of PilR/PilS in the regulation of pilin production and binding to collagen by comparison of isogenic pilR-null and complemented strains with the parent strain CP1. In addition, the role of the Agr-like QS system in regulating pilin production and binding has been examined by comparing isogenic agrB-null and virR-null mutants and their respective complemented strains with CP1. Western blot analyses showed no detectable pilus production in mutant pilR, while production by its complemented strain was similar to wild-type levels. In contrast, pilus production was the same or higher in the agrB and virR mutants but reduced in their respective complements compared with CP1. Similarly, no pilus production was detected in the pilin mutant strains (cnaA, fimA, or fimB). These observations were supported by the binding results to collagen of mutants pilR, agrB, and virR and their complemented strains, with CP1 as a reference. The pilR mutant showed significantly less binding than CP1 (P ≤ 0.05) to most collagen types, but its complement was similar to the parent strain in binding (P > 0.05). In contrast, binding of agrB and virR mutants to collagen (types I – V) showed no significant changes compared with CP1 (P > 0.05), whereas their complemented strains had significantly reduced binding to collagen types I – IV (P ≤ 0.05). To confirm that the collagen binding activity of the agrB mutant was specifically mediated by the pilus, and not due to the expression of other factors regulated by the Agr-like QS system, binding of CP1 and mutant agrB to type I and IV collagen was examined in the presence of anti-CnaA, FimA, or FimB antibodies. Antibodies against both CnaA and FimA blocked the binding of CP1 and the mutant in a dose-dependent manner. However, the antibody against FimB showed no blocking of binding. These data suggest that the PilR/PilS two-component system positively regulates pilin production, while the Agr-like QS system may negatively regulate pilin production in C. perfringens.