Evidence That VirS Is a Receptor for the Signaling Peptide of the Clostridium perfringens Agr-like Quorum Sensing System

Abstract

Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics. IMPORTANCE C. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections.

Pathogenicity and virulence of Clostridium perfringens

“…Computer modeling predicts that the VirS protein consists of seven predicted transmembrane domains, several exposed extracellular regions, and a C-terminal tail [ 161 , 162 ]. The C-terminal tail is located in the cytoplasm and contains several conserved motifs typical of histidine kinases, including the likely site of autophosphorylation, i.e.…”

“…H255 and the G box, which is involved in ATP binding [ 161 ]. A recent study [ 162 ] implicated the 2 nd extracellular loop of VirS in signal sensing (see quorum sensing section). When this signal is received, VirS autophosphorylates and then activates VirR by transferring a phosphate onto a conserved aspartate residue located in the N-terminal region of VirR [ 161 , 162 ].…”

“…A recent study [ 162 ] implicated the 2 nd extracellular loop of VirS in signal sensing (see quorum sensing section). When this signal is received, VirS autophosphorylates and then activates VirR by transferring a phosphate onto a conserved aspartate residue located in the N-terminal region of VirR [ 161 , 162 ]. The C-terminal domain of activated VirR recognizes and binds to VirR boxes, which are two imperfect, directly repeated sequences located upstream of the target gene [ 163 , 164 ].…”

“…However, the C. perfringens agr operon does not encode an AgrA/AgrC homolog. Since the production of several C. perfringens toxins, including PFO, CPA, CPB, and NetB, is co-regulated by both the Agr-like QS and VirS/VirR TCRS, it was hypothesized that the VirS membrane sensor protein is an AIP receptor for the C. perfringens Agr-like QS system [ 177 ]; this proposal was recently confirmed when the 2 nd extracellular loop of VirS was shown to be involved in AIP binding [ 162 ]. A model for the interactions between the Agr-like QS and VirS/VirR TCRS is depicted in Figure 3 .…”

Reevaluation of whether a Functional Agr-like Quorum-Sensing System Is Necessary for Production of Wild-Type Levels of Epsilon-Toxin by Clostridium perfringens Type D Strains

“…Since the publication of that 2011 paper, the reliability of concluding that the Agr QS is necessary for production of wild-type ETX levels has come into question. Specifically, it has since been shown that (i) neither the Agr QS system nor the VirS VirR two-component regulatory system (TCRS) is required for production of wild-type ETX levels by two type B strains ( 6 ), (ii) production of all other C. perfringens toxins regulated by the Agr QS system also involves the VirS VirR TCRS ( 7 – 14 ), and (iii) the Agr QS signal peptide can bind directly to VirS as a receptor ( 15 ).…”