抗菌肽F1对金黄色葡萄球菌的胞内作用机制

Intracellular Mechanism of Action of Antimicrobial Peptide F1 on Staphylococcus aureus

研究副干酪乳杆菌FX-6产抗菌肽F1对金黄色葡萄球菌胞内抑菌作用机制。通过凝胶阻滞电泳、荧光光谱考察抗菌肽F1对金黄色葡萄球菌DNA的结合作用及方式,考马斯亮蓝法检测、聚丙烯酰胺凝胶电泳考察抗菌肽F1对金黄色葡萄球菌蛋白合成的影响,邻硝基苯-β-D-吡喃半乳糖苷法、荧光指示剂法分别测定抗菌肽F1对金黄色葡萄球菌胞内β-半乳糖苷酶、非特异酯酶表达活性的影响,流式细胞仪分析F1对金黄色葡萄球菌细胞周期的影响。结果显示,抗菌肽F1能以嵌插方式与金黄色葡萄球菌细胞内的DNA结合,阻碍其遗传信息正常表达,使其蛋白的合成减少,并使胞内β-半乳糖苷酶、非特异性酯酶的表达活性减弱,胞内酶系统紊乱,影响其正常代谢,生长周期无法顺利进行,从而起到抑菌的效果。

This study aimed to elucidate the intracellular mechanism of action of antimicrobial peptide F1on Staphylococcusaureus.The binding of the genomic DNA of S.aureus to antimicrobial peptide F1was investigated with gel retardation,and the competitive intercalation of F1and ethidium bromide(EB)into the genomic DNA were analyzed by fluorescencespectroscopy.The influence of F1on the synthesis of protein in S.aureus was determined by Coomassie brilliant blue methodand sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The inhibition ofβ-galactosidase and nonspecificesterase activities of S.aureus cells by the antibacterial peptide was measured using o-nitrophenylβ-D-galactopyranosiden(ONPG)and di-O-acetylfluorescein(FDA),respectively.Finally,the changes in the cell cycle of S.aureus after being treated by F1were monitored with flow cytometry.The results showed that F1bound with DNA and could weaken the fluorescence intensity ofEB-DNA complex,hindering the expression of genetic information of S.aureus.Besides,the protein biosynthesis of S.aureus wasalso inhibited,which was further proven by the measured activities of two intracellular enzymes,β-galactosidase and non-specificesterase.Therefore,F1could lead to the aberration of the intracellular enzyme system and metabolic pathways of S.aureus,andblock the DNA synthesis phase of the cell cycle of S.aureus.