[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.
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