基于分子对接的副溶血弧菌外膜蛋白OmpW天然抑制剂的筛选

Screening of Natural Inhibitors of Outer Membrane Protein OmpW of Vibrio parahaemolyticus Based on Molecular Docking

为了筛选更加高效、无毒、绿色的副溶血弧菌抑制剂,以副溶血弧菌外膜蛋白OmpW作为受体,利用分子对接方法筛选具有潜在抑菌作用的天然小分子,选用对接结果较理想的柠檬酸和咖啡酸进行试验验证。在测定最小抑菌浓度的基础上,研究去外膜和去壁的副溶血弧菌对小分子敏感性及杀菌曲线的变化,分析外膜蛋白OmpW是否为柠檬酸和咖啡酸的作用位点,验证基于分子对接筛选细菌抑制剂方法的正确性。研究结果显示,柠檬酸与咖啡酸对副溶血弧菌ATCC17802的最小抑制浓度均为2 mg/mL。去外膜和去壁的菌体与对照相比,最小抑制浓度减小16~64倍,这表明柠檬酸和咖啡酸很有可能作用于副溶血弧菌的外膜蛋白OmpW,分子对接结果也显示二者通过非共价结合发挥抑制作用。验证性试验表明:这两种小分子对于副溶血弧菌其它菌株类型也有同样的抑制效果。结论:柠檬酸和咖啡酸是有效的副溶血弧菌抑制剂(最小抑制浓度<5 mg/mL),其作用位点很可能是外膜蛋白OmpW。利用分子对接方法寻找高效、绿色的细菌抑制剂具有广阔的发展前景。

In order to screen more efficient,non-toxic and green inhibitors of Vibrio parahaemolyticus,molecular docking was applied to screen natural small molecules with potential antimicrobial activity using the outer membrane protein OmpW of V.parahaemolyticus as receptor.Then citric acid and caffeic acid which had ideal docking results were used for further experimental verification.On the basis of determining the minimal inhibitory concentrations(MIC),the changes of sensitivity to small molecules and the killing curves of V.parahaemolyticus with outer membrane removed and cell wall removed were studied,and whether outer membrane protein OmpW was the binding site of citric acid and caffeic acid was analyzed to verify the correctness of the method based on molecular docking for screening bacterial inhibitors.The results showed that the MICs of citric acid and caffeic acid on V.parahaemolyticus ATCC17802 were both 2 mg/mL.Compared with the control,the MICs of the cells with outer membrane removed and cell wall removed were reduced by 16 to 64 times,which indicated that citric acid and caffeic acid were likely to act on outer membrane protein OmpW of V.parahaemolyticus,and molecular docking results also indicated that these two small molecules formed non-covalent binding with OmpW to fulfill inhibition.Validation tests showed that these two small molecules also restrained other strains of V.parahaemolyticus.Conclusion:citric acid and caffeic acid are effective inhibitors of V.parahaemolyticus(MIC<5 mg/mL),and their action site is likely to be the outer membrane protein OmpW.The use of molecular docking to search for efficient and green bacterial inhibitors has a broad development prospect.