Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, Australia.
Institute for Glycomics, Griffith University, Gold Coast 4222, Australia.
Int J Med Microbiol. 2020 Feb;310(2):151398. doi: 10.1016/j.ijmm.2020.151398. Epub 2020 Jan 17.
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.
产气荚膜梭菌是人类气性坏疽的病原体；与该病相关的主要毒素是α-毒素和产气荚膜梭菌溶细胞素 O。RevSR 双组分调控系统已被证明参与调控小鼠肌坏死模型中的毒力。先前对 revR 突变体的微阵列和 RNAseq 分析表明，主要毒素以外的因素可能在毒力中发挥作用。RNAseq 数据显示，编码 EngCP 内切 α-N-乙酰半乳糖胺酶（CPE0693）的基因表达在 revR 突变体中显著下调。该家族的酶已在几种革兰氏阳性病原体中被鉴定出来，并被推测有助于它们的毒力。在本研究中，我们构建了产气荚膜梭菌的 engCP 突变体，并表明其毒力明显低于野生型亲本菌株。通过用野生型 engCP 基因在体外互补恢复了毒力。我们还证明了纯化的 EngCP 能够水解来自 C2C12 小鼠肌管的α-肌营养不良蛋白。然而，EngCP 对小鼠的膜通透性几乎没有影响，这表明 EngCP 可能发挥了除破坏肌纤维结构完整性以外的作用。糖基阵列分析表明，EngCP 可以识别含有单糖 N-乙酰半乳糖胺的结构，在 EngCP 具有酶活性的条件下，它可以识别以半乳糖、葡萄糖和 N-乙酰葡萄糖胺结尾的结构。总之，我们已经获得了证据表明 EngCP 是产气荚膜梭菌毒力所必需的，尽管经典外毒素对疾病很重要，但我们现在已经表明，一种 O-糖苷酶也在疾病过程中发挥着重要作用。
