绿原酸体内代谢产物抗金黄色葡萄球菌生物被膜作用及机制研究

Inhibitory Effects of Chlorogenic Acid Metabolites on Biofilm of Staphylococcus Aureus and Its Mechanism

目的:研究绿原酸三种体内代谢物苯甲酸、对香豆酸和对羟基苯甲酸对耐甲氧西林金黄色葡萄球菌(MRSA)生物被膜的作用及作用机制。方法:微量肉汤稀释法测定苯甲酸、对香豆酸和对羟基苯甲酸对MRSA最低抑菌浓度(MIC),并用棋盘稀释法测定联合抑菌浓度指数(FIC),结晶紫染色法观察三者对MRSA生物被膜的影响;荧光定量PCR法检测样品对细菌主要致病基因Agra、Icaa、Icad、Icab的水平,并通过分子对接法评价三者与MRSA生物被膜关键蛋白AgrA活性位点的竞争性结合。结果:对香豆酸和对羟基苯甲酸对MRSA的抑菌活性较绿原酸有所增强;苯甲酸与对羟基苯甲酸、对香豆酸与对羟基苯甲酸联用产生抑菌协同增强作用;在亚抑菌浓度下,与正常对照组相比,三种代谢产物对生物被膜形成均有明显抑制作用,对成熟生物被膜均有明显消除作用(P<0.05或P<0.01);苯甲酸在0.50 mg/mL~0.12 mg/mL,对香豆酸、对羟基苯甲酸在0.25 mg/mL~0.06 mg/mL浓度范围对Agra基因均明显下调,对Icaa基因明显上调(P<0.05或P<0.01);分子对接结果显示,三者与AgrA蛋白均有一定亲和力,其中对香豆酸亲和力较强,对AgrA蛋白功能影响更大。结论:绿原酸体内主要代谢物苯甲酸、对香豆酸和对羟基苯甲酸的抗菌作用较绿原酸更强,部分在体内发挥协同抗菌增强作用;三种代谢产物在一定亚抑菌浓度范围内抑制和消除MRSA生物被膜;代谢产物通过与AgrA蛋白的竞争性结合,调控相关致病基因表达,降低细菌致病性。

Objective: To investigate the inhibitory effects and mechanism of three main metabolites of chlorogenic acid on the biofilm of methicillin-resistant Staphylococcus aureus(MRSA), including benzoic acid, p-coumaric acid, and p-hydroxybenzoic acid. Methods: The minimum inhibitory concentrations(MICs) of benzoic acid, p-coumaric acid, and p-hydroxybenzoic acid to MRSA were tested by microtiter broth dilution method.Fractional inhibitory concentration(FIC) was measured by checkerboard dilution method. Crystal violet staining was used to observe the effects on MRSA biofilm. The levels of major pathogenic genes including Agra, Icaa, Icad, and Icab were measured by fluorescence-based quantitative polymerase chain reaction(PCR). Molecular docking analysis was used to observe the competitive binding with the active site of key protein AgrA in the biofilm. Results: The antibacterial activities of p-coumaric acid and p-hydroxybenzoic acid on MRSA were potentiated as compared with that of chlorogenic acid. Benzoic acid combined with p-hydroxybenzoic acid and p-coumaric acid combined with p-hydroxybenzoic acid showed enhanced synergistic antibacterial effects. At sub-inhibitory concentrations, compared with the normal control group, the three metabolites showed inhibitory effects on biofilm formation and elimination effects on mature biofilms(P<0.01 or P<0.05). Benzoic acid(0.50-0.12 mg/mL), p-coumaric acid(0.25-0.06 mg/mL), and p-hydroxybenzoic acid(0.25-0.06 mg/mL) significantly down-regulated Agra gene and up-regulated Icaa gene(P<0.01 or P<0.05). Molecular docking results revealed that the three metabolites showed certain affinities with AgrA protein, especially p-coumaric acid, which had a great effect on the function of AgrA protein. Conclusion: The three metabolites of chlorogenic acid, i.e., benzoic acid, p-coumaric acid, and p-hydroxybenzoic acid, possess strong antibacterial effects than chlorogenic acid, and some metabolites show enhanced synergistic antibacterial effects in vivo. At sub-inhibitory concentrations, the three metabolites can inhibit and eliminate the MRSA biofilm. The metabolites regulate the expression of related pathogenic genes and reduce bacterial pathogenicity by competitively binding with AgrA protein.