一种源自人α-2-巨球蛋白的抗菌肽A2M3及其对金黄色葡萄球菌的抑菌机制  

A novel antimicrobial peptide A2M3 derived from human alpha-2-macroglobulin inhibits Staphylococcus aureus

作　　者：范学楠 林蓉 金日天 梁铎 邱绪健[1,3] 杨燊 FAN Xuenan;LIN Rong;JIN Ritian;LIANG Duo;QIU Xujian;YANG Shen(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,Fujian,China;Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering,Jimei University,Xiamen 361021,Fujian,China;Collaborative Innovation Center for Key Technologies of Deep Processing of Marine Food,Dalian Polytechnic University,Dalian 116034,Liaoning,China)

机构地区：[1]集美大学海洋食品与生物工程学院,福建厦门361021 [2]集美大学福建省食品微生物与酶工程重点实验室,福建厦门361021 [3]大连工业大学,海洋食品精深加工关键技术省部共建协同创新中心,辽宁大连116034

出　　处：《微生物学报》2024年第3期938-952,共15页Acta Microbiologica Sinica

基　　金：福建省高校产学合作项目(2023N5008);厦门市科技补助项目(2023CXY0305)。

摘　　要：【目的】在人的鼻腔中鉴定出一种源自α-2-巨球蛋白的抗菌肽(命名为A2M3),并探究其对金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用和机制。【方法】结合生物信息学技术对人类鼻液的质谱结果进行分析,并筛选潜在抗菌肽;通过微量稀释法和平板涂布法分别分析A2M3对金黄色葡萄球菌最低抑菌浓度(minimum inhibitory concentration,MIC)和时间杀伤曲线(time-kill curve);采用透射电镜、碘化丙锭(propidium iodide,PI)摄取实验、流式细胞术和核酸蛋白质泄露实验分析A2M3对金黄色葡萄球菌膜完整性、膜通透性的影响;通过凝胶阻滞实验和荧光光谱实验探究A2M3对金黄色葡萄球菌基因组DNA的影响。【结果】利用生物信息学技术筛选出源自α-2-巨球蛋白的潜在抗菌肽A2M3,其对金黄色葡萄球菌的MIC为125.0μg/mL,且能在3h内完全杀灭细菌。A2M3通过增加细胞膜的通透性,促使核酸和蛋白质泄漏,继而穿过细胞膜嵌入DNA的碱基对,影响细菌的基因功能,从而导致菌体死亡。【结论】A2M3对金黄色葡萄球菌的抑菌机制涉及多靶点协同作用,能够改变细菌细胞膜的通透性,影响细菌的基因功能。这一发现揭示了从人体体液中筛选和分离抗菌功能肽的潜在应用价值。[Objective]To study the inhibitory effect and mechanism of an antimicrobial peptide(A2M3)derived from alpha-2-macroglobulin identified in the human nasal cavity against Staphylococcus aureus.[Methods]The mass spectrometry results of the human nasal liquid were analyzed,on the basis of which bioinformatic tools were used for the screening of potential antimicrobial peptides.The minimum inhibitory concentration(MIC)and time-kill curve of A2M3 against S.aureus were determined by the microdilution method and plate colony counting method.Then,transmission electron microscopy,PI uptake assay,flow cytometry,and determination of nucleic acid protein leakage were employed to study the effects of A2M3 on the membrane integrity and permeability of S.aureus.Finally,the effect of A2M3 on the genomic DNA of S.aureus was investigated by the gel retardation assay and fluorescence spectroscopy.[Results]A2M3 showed an MIC of 125.0μg/mL against S.aureus and killed the bacteria completely within 3 h.A2M3 increased the cell membrane permeability to penetrate into S.aureus cells,leading to leakage of nucleic acids and proteins as well as insertion into DNA base pairs to interfere with the gene function,resulting in the death of the cells.[Conclusion]The inhibitory mechanism of A2M3 against S.aureus involves multiple targets.The antimicrobial peptide alters the permeability of the bacterial cell membrane and affects the gene function,thus exerting the inhibitory activity.The findings reveal that antimicrobial peptides could be screened and isolated from human body fluids for potential application.

关 键 词：鼻液 α-2-巨球蛋白 金黄色葡萄球菌 抗菌肽 抑菌机制 

分 类 号：R378.11[医药卫生—病原生物学]