School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou, 510006, China.
Appl Microbiol Biotechnol. 2022 Jun;106(12):4511-4521. doi: 10.1007/s00253-022-12010-0. Epub 2022 Jun 14.
Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding domains, proven to be involved in bacterial cell wall remodeling and cell division. Although autolysins in C. difficile have been reported, the autolysins have failed to yield impressive results when used as exogenous lytic agents. In this study, we expressed and characterized the binding domains (Cwp19-BD and Acd-BD) and catalytic domains (Cwp19-CD, Acd-CD, and Cwl-CD) of C. difficile autolysins, and the domains with the best binding specificity and lytic activity were selected towards C. difficile to design a novel lytic protein Cwl-CWB2. Cwl-CWB2 showed good biosafety with significantly low hemolysis and without cytotoxicity. The results of fluorescence analysis and lytic assay demonstrated that Cwl-CWB2 has higher binding specificity and stronger lytic activity with a minimum inhibitory concentration at 13.39 ± 5.80 μg/mL against living C. difficile cells, which is significantly stronger than commercial lysozyme (3333.33 ± 1443.37 μg/mL) and other reported C. difficile autolysins. Besides, Cwl-CWB2 exhibited good stability as about 75% of the lytic activity was still retained when incubated at 37 °C for 96 h, which is considered to be a potential antimicrobial agent to combat C. difficile. KEY POINTS: • Several binding domains and catalytic domains, deriving from several Clostridium difficile autolysins, were expressed, purified, and functionally characterized. • A novel C. difficile lytic protein Cwl-CWB2 was designed from C. difficile autolysins. • The binding specificity and lytic activity of Cwl-CWB2 against C. difficile showed advantages compared with other reported C. difficile autolysins. • Cwl-CWB2 exhibited significantly low hemolysis and cytotoxicity against normal-derived colon mucosa 460 cell.
艰难梭菌（C. difficile）是一种革兰氏阳性、产芽孢、产毒的厌氧菌，可引起医院获得性抗生素相关性肠道疾病。自溶素是一种裂解酶，可水解细菌细胞壁的肽聚糖，具有催化结构域和细胞壁结合结构域，已被证明参与细菌细胞壁重塑和细胞分裂。尽管已经报道了艰难梭菌中的自溶素，但当用作外源性裂解剂时，自溶素并未产生令人印象深刻的结果。在这项研究中，我们表达和表征了艰难梭菌自溶素的结合结构域（Cwp19-BD 和 Acd-BD）和催化结构域（Cwp19-CD、Acd-CD 和 Cwl-CD），并选择了具有最佳结合特异性和裂解活性的结构域来设计一种新型的裂解蛋白 Cwl-CWB2。Cwl-CWB2 具有良好的生物安全性，溶血率低，无细胞毒性。荧光分析和裂解测定的结果表明，Cwl-CWB2 对活的艰难梭菌细胞具有更高的结合特异性和更强的裂解活性，最低抑制浓度为 13.39 ± 5.80 μg/mL，明显强于商业溶菌酶（3333.33 ± 1443.37 μg/mL）和其他报道的艰难梭菌自溶素。此外，Cwl-CWB2 表现出良好的稳定性，在 37°C 孵育 96 h 时，约 75%的裂解活性仍得以保留，被认为是一种有潜力的抗艰难梭菌的抗菌剂。 关键点： • 表达、纯化并功能表征了来自几种艰难梭菌自溶素的几种结合结构域和催化结构域。 • 从艰难梭菌自溶素设计了一种新型的艰难梭菌裂解蛋白 Cwl-CWB2。 • Cwl-CWB2 对艰难梭菌的结合特异性和裂解活性与其他报道的艰难梭菌自溶素相比具有优势。 • Cwl-CWB2 对正常来源的结肠黏膜 460 细胞的溶血率和细胞毒性明显较低。
