NDM-5-C208A突变大肠埃希菌的构建及大蒜辣素与NDM-5相互作用位点分析  

Construction of NDM-5-C208A mutant Escherichia coli as well as the analysis of interaction between allicin and NDM-5

作　　者：叶志滨 王晓明 吕茜 刘雨桐 金季赜晓 朱馨艺 黄金虎[1] 王丽平[1] YE Zhibin;WANG Xiaoming;Lü Xi;LIU Yutong;JINJI Zexiao;ZHU Xinyi;HUANG Jinhu;WANG Liping(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2024年第1期61-68,共8页Journal of Nanjing Agricultural University

基　　金：江苏省自主创新项目(CX[22]3039)。

摘　　要：[目的]产新德里金属β-内酰胺酶5(NDM-5)的革兰阴性耐药菌对人畜健康产生巨大威胁。本文旨在初步探究大蒜辣素对NDM-5的抑制作用及其相关作用位点,为临床开发新型β-内酰胺酶抑制剂提供理论支持。[方法]通过AutoDock模拟分子对接,预测大蒜辣素与NDM-5的结合位点;构建突变型载体pET21a-NDM-5-C208A,测序验证无误后转入感受态大肠埃希菌BL21(DE3),通过药敏试验进行突变表型确认;采用微量肉汤棋盘法和时间杀菌曲线测定大蒜辣素与美罗培南对NDM-5突变型细菌的联合抑菌效果。通过体外表达突变蛋白NDM-5-C208A,建立酶活性测定体系,比较大蒜辣素对C208突变型NDM-5和野生型NDM-5酶活性的抑制效果。[结果]经测序证实NDM-5的C208A突变株BL21(DE3)pET21a-NDM-5-C208A构建成功,C208A突变株恢复了对头孢类和碳青霉烯类抗生素的敏感性,且大蒜辣素与美罗培南联用的联合抑菌指数由0.375增加至2,联用表现为独立作用而非协同作用;体外酶活测定结果显示NDM-5-C208A突变不仅使酶活性下降约85%,而且该位点突变导致大蒜辣素对NDM-5失去抑制作用。[结论]Cys208位点是维持NDM-5水解酶活性的关键位点,且大蒜辣素可通过与该位点结合发挥抑制酶活性的作用。该结果为大蒜辣素作为新型β-内酰胺酶抑制剂的开发和应用提供试验依据。[Objectives]Gram-negative resistant bacteria producing New Delhi metallo-β-lactamase-5(NDM-5)pose great threat to the health of human and domestic animals.The study aimed to explore the inhibitory action and the involved mechanism of allicin on NDM-5,which could provide theoretical support for the development of novel β-lactamase inhibitors.[Methods]The binding site of allicin and NDM-5 was predicted by molecular docking with AutoDock.Mutant vector pET21a-NDM-5-C208A was constructed and transferred into E.coli BL21(DE3)competent cell after sequencing.Sensitive test was performed to verify the phenotype of mutant strain.Checkerboard method and time-kill curves assay were performed to assess the combination effect of allicin and meropenem against NDM-5 mutant strain.Enzyme activity assay was established to compare the inhibitory effect of allicin on the activities of C208 mutant NDM-5 and wild-type NDM-5 by expressing the mutant protein NDM-5-C208A in vitro.[Results]The C208A mutant strain BL21(DE3)pET21a-NDM-5-C208A was successfully constructed confirmed by sequencing.The C208A mutant strain regained susceptibility to cephalosporins and carbapenems,and the combination of allicin and meropenem showed independent effect,but not synergistic effect(FICI increased from 0.375 to 2).Enzyme activity assay showed that C208A mutation of NDM-5 caused the decrease of enzyme activity by 85% in vitro and the mutation resulted in the loss of inhibitory effect of allicin on NDM-5.[Conclusions]The Cys208 is a key site for maintaining the enzymatic hydrolysis activity of NDM-5,and allicin can exert an inhibitory effect on the enzyme activity by binding to this site.The results provide a strong support for developing and applying of allicin as a novel β-lactamase inhibitor.

关 键 词：大蒜辣素 NDM-5 美罗培南 酶抑制剂 

分 类 号：S852.6[农业科学—基础兽医学]