金抗肽SIF_(4)基于糖代谢途径和细胞质膜氧化损伤的大肠埃希菌抑菌机理

Antimicrobial mechanism of metal antimicrobial peptide SIF_(4) against Escherichia coli based on glucose metabolism pathway and cytoplasmic membrane oxidative damage

为揭示金抗肽SIF_(4)基于糖代谢途径和细胞质膜氧化损伤的大肠埃希菌抑菌机理,研究了SIF_(4)对糖酵解和三羧酸循环途径关键酶活性、细胞质膜氧化损伤及细胞内源活性氧自由基(reactive oxygen species,ROS)生成的影响。研究发现,SIF_(4)对己糖激酶、磷酸果糖激酶和丙酮酸激酶这3种糖酵解关键酶抑制率分别达66.61%、27.37%和39.61%,对丙酮酸脱氢酶系、柠檬酸合成酶、异柠檬酸脱氢酶和α-酮戊二酸脱氢酶这4种三羧酸循环关键酶抑制率分别达25.20%、22.91%、26.99%和23.44%,主要通过抑制大肠埃希菌糖酵解途径中已糖激酶活性来调控葡萄糖氧化效率而发挥抑菌活性。研究还发现,SIF_(4)可造成细胞质膜氧化损伤和诱导氧化应激产生过量ROS,破坏细胞质膜结构与功能,从而诱导菌体细胞凋亡或程序性死亡来发挥协同抑菌活性。试验结果可为系统揭示SIF_(4)对大肠埃希菌抑菌机理提供直接证据,也为其在食源性大肠埃希菌生物防控中的应用提供理论支持。

To reveal the antimicrobial mechanism of metal antimicrobial peptide(MAP)SIF_(4) against Escherichia coli based on glucose metabolism pathway and cytoplasmic membrane oxidative damage,the effects of SIF_(4) on critical enzymatic activity of Embden-Meyerhof-Parnas(EMP)pathway and tricarboxylic acid(TCA)cycle,cytoplasmic membrane oxidative damage,intracellular ROS production and cell membrane depolarization were investigated.Results showed that,SIF_(4) inhibited the key enzymes in glycolysis,including hexokinase(HK),phosphofructokinase(PFK)and pyruvate kinase(PK),by 66.61%,27.37,and 39.61%,respectively,and the key enzymes in TCA cycle,including pyruvate dehydrogenase complex(PDHC),citrate synthetase(CS),isocitrate dehydrogenase(IDH)and α-ketoglutaric dehydrogenase(α-KGDH),by 25.20%,22.91%,26.99 and 23.44%,respectively.SIF_(4) exerted good antimicrobial capacity mainly through regulating glucose oxidation efficiency by restraining the HK activity in glycolysis.It was also confirmed that SIF_(4) could cause cytoplasmic membrane oxidative damage and induce oxidative stress to produce excessive ROS,resulting in destroying cytoplasmic membrane structural integrity,and exerting synergistic enhancement of antimicrobial capacity by inducing cell apoptosis or programmed cell death.All results can provide direct evidence for systematically elucidating antimicrobial mechanism of SIF_(4) against E.coli,and also offer theoretical support for its application in the biological control of foodborne E.coli.