Determination of guaifenesin and dextromethorphan in a cough syrup by HPLC with fluorometric detection

Abstract

Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of <i>Clostridium perfringens</i> positively regulate production of several toxins, including <i>C. perfringens</i> 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 <i>virS</i> gene in <i>C. perfringens</i> strains CN3685 and CN1795 because it was reported that <i>agrB</i> mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 <i>agrB</i> 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, <i>virS agrB</i> 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 <i>agrB</i> 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.<b>IMPORTANCE</b><i>C. perfringens</i> 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 <i>C. perfringens</i> 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 <i>C. perfringens</i> pathogenicity and may provide insights for designing novel strategies to reduce <i>C. perfringens</i> toxin production during infections.