Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

产气荚膜梭状芽胞杆菌是人类梭菌性肌坏死的病原体。该疾病涉及的主要毒素是α毒素和穿孔蛋白溶酶O。RevSR两组分调节系统已显示参与调节小鼠心肌坏死模型的毒力。以前对revR突变体进行的微阵列和RNAseq分析表明，除主要毒素外，其他因素也可能在毒力中起作用。的RNA测序数据表明，该基因的编码内切EngCP的α-表达Ñ -acetylgalactosaminidase（CPE0693）是一个被显著下调REVR突变体。该家族的酶已在几种革兰氏阳性病原体中鉴定出来，并被认为有助于它们的毒性。在这项研究中，我们构建了产气荚膜梭菌的engCP突变体，并表明其毒性远低于其野生型亲本菌株。通过与野生型engCP反式互补来恢复毒力基因。我们还证明了纯化的EngCP能够水解衍生自C2C12小鼠肌管的α-dystroglycan。但是，EngCP对小鼠的膜通透性几乎没有影响，这表明EngCP可能除了破坏肌纤维的结构完整性以外，还可能发挥其他作用。聚糖阵列分析表明，EngCP可以识别包含所述单糖结构Ñ在4℃-acetlygalactosamine，但可以识别结构半乳糖，葡萄糖和终止Ñ条件下乙酰氨基葡萄糖，其中EngCP是酶促活性。总而言之，我们已经获得了证据，证明EngCP是产气荚膜梭菌的毒力所必需的，尽管经典的外毒素对疾病很重要，但我们现在已经证明O-糖苷酶在疾病过程中也起重要作用。