南京农业大学学报2024，Vol.47Issue(1)：61-68,8.DOI:10.7685/jnau.202210009
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
摘要
Abstract
[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Key words
allicin/NDM-5/meropenem/enzyme inhibitor分类
农业科技引用本文复制引用
叶志滨,王晓明,吕茜,刘雨桐,金季赜晓,朱馨艺,黄金虎,王丽平..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[J].南京农业大学学报,2024,47(1):61-68,8.基金项目
江苏省自主创新项目(CX[22]3039) （CX[22]3039）
