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.

中文翻译：

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VirS是产气荚膜梭菌Agr样群体感应系统的信号肽受体的证据。

由于类似Agr（辅助基因调节剂）的群体感应（QS）系统和VirS / VirR（VirS / R）产气荚膜梭菌的两组分调节系统都可以积极调节多种毒素的产生，包括产气荚膜梭菌β毒素（CPB） ，已经假设VirS膜传感器蛋白是一种Agr样QS信号肽（SP）受体。为了开始评估VirS是否为SP受体，本研究对产气荚膜梭菌菌株CN3685和CN1795中的virS基因进行了测序，因为据报道，这两种菌株的agrB突变体均响应五肽5R（可能是天然SP）增加了CPB的产量，但只有CN3685 agrB突变体对8R作出反应，这是5R加上3个氨基酸的尾巴。该测序鉴定出CN3685的预测VirS细胞外环2（ECL2）与CN1795的预测VirS细胞外环2之间的差异。为了探讨这些ECL2差异是否解释了菌株敏感性和SP相关性，并支持VirS作为SP受体，对每个菌株的virS agrB双无效突变体进行了互补，以交换它们产生的VirS蛋白。CPB蛋白质印迹表明，这种互补作用将每种菌株的自然反应性更改为8R。使用生物素5R的下拉实验证明VirS可以结合SP。为了进一步支持VirS：SP结合并鉴定SP的VirS结合位点，合成了对应于VirS ECL2的14-mer肽。该ECL2肽抑制了向agrB的5R信号转导突变株和野生株。这种抑制作用是特异性的，因为ECL2肽中的单个N到D取代消除了这些作用。总的来说，这些结果支持VirS作为重要的SP受体，并可能有助于治疗剂的开发。