抗菌肽-Mt6的理化特性和抗菌活性及联合抗真菌药物对白色念珠菌的作用  

Physicochemical properties and antimicrobial activity of antimicrobial peptide Mt6 and combined antifungal effect against andida albicans

作　　者：黄敏慧 曾晔 张迎春 李彩多 吴建伟 陈峥宏 王涛 HUANG Minhui;ZENG Ye;ZHANG Yingchun;LI Caiduo;WU Jianwei;CHEN Zhenghong;WANG Tao(School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]贵州省普通高等学校病原生物学特色重点实验室,贵州贵阳550025

出　　处：《贵州医科大学学报》2023年第6期627-633,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81360254);贵州省科技计划项目(黔科合平台人才[2018]5799-22,黔科合支撑[2020]4Y236)。

摘　　要：目的探讨抗菌肽-Mt6(AMPs-Mt6)的理化特性、抗菌活性及联合抗真菌药物对白色念珠菌(C.albicans)的作用。方法以家蝇抗真菌肽-1A(MAF-1A)为模板,采用氨基酸残基替换法设计AMPs-Mt6,并以生物信息学方法分析其理化特性;挑取对数生长期C.albicans、金黄色葡萄球菌(S.aureus)、枯草杆菌(B.subtilis)、大肠埃希菌(E.coli)、铜绿假单胞菌(P.aeruginosa)、甲型副伤寒沙门菌(S.paratyphi)及肺炎克雷伯菌(K.pneumoniae)菌落制备细胞悬液,取菌液100μL和相同体积不同终浓度(156.25~2500.00 mg/L)的AMPs-Mt6稀释液混匀,采用微量肉汤稀释法检测AMPs-Mt6对上述各菌属的最小抑菌浓度(MIC)、最小杀真菌浓度(MFC)及最小杀细菌浓度(MBC);取(0.5~2.5)×10^(6)cfu/L的C.albicans菌液100μL和相同体积不同终浓度(0.25~64.00 mg/L)的氟康唑(FLC)、两性霉素B(AmB)、伊曲康唑(ITC)及5-氟胞嘧啶(5-FC)药物稀释液混匀,采用微量肉汤稀释法检测上述各药物对C.albicans的MIC和MFC;采用棋盘格法检测AMPs-Mt6分别与FLC、AmB、ITC、5-FC联合使用时对C.albicans的体外抑制活性,并以部分抑菌浓度指数(FIC index)判定联合效应。结果AMPs-Mt6为带有9个正电荷数、疏水性为0.451、α-螺旋占比81%的两亲性多肽;AMPs-Mt6对C.albicans的MIC为312.50 mg/L,对所检测细菌均有抗菌作用,对S.aureus、B.subtilis及P.aeruginosa的MIC为625.00 mg/L,对E.coli、S.paratyphi、K.pneumoniae的MIC为1250.00 mg/L;AMPs-Mt6与AmB、ITC及5-FC联合时,FIC index值分别为0.19、0.36及0.37,表现为协同效应;AMPs-Mt6与FLC联合时,FIC index值为0.63,为相加效应。结论与模板肽MAF-1A相比,AMPs-Mt6的理化性质得到优化,抗菌活性和抗菌谱增强,与抗真菌药FLC、AmB、ITC及5-FC联用可增强各药物对C.albicans的抑杀效果。Objective To construct a novel antimicrobial peptide Mt6,and investigate physicochemical properties and antimicrobial activity of antimicrobial peptide Mt6(AMPs-Mt6),and in vitro combined effect against Candida albicans(C.albicans)with antifungal agents.Methods AMPs-Mt6 was modified by amino acid residue replacement method,and its physicochemical properties were analyzed by bioinformatics method,with Musca domestica antifungal peptide-1A(MAF-1A)as the modification template.The colonies from logarithmic phase cultures of C.albicans,Staphylococcus aureus(S.aureus),Bacillus subtilis(B.subtilis),Escherichia coli(E.coli),Pseudomonas aeruginosa(P.aeruginosa),Salmonella paratyphi(S.paratyphi),Klebsiella pneumoniae(K.pneumoniae.)were picked and prepared into cell suspension;the same volume of 100μL of bacteria suspension and AMPs-Mt6 dilution(156.25 to 2500.00 mg/L)of different final concentration were mixed.The minimum inhibitory concentration(MIC),minimum fungicidal concentration(MFC)and minimum bactericidal concentration(MBC)of AMPs-Mt6 against the above-mentioned bacteria and fungus were detected by microbroth dilution method.100μL of C.albicans solution of(0.5-2.5)×10^(6) cfu/L was mixed with diluents of Fluconazole(FLC),Amphotericin B(AmB),Itraconazole(ITC),and 5-Fluorocytosine(5-FC)in the same volume and at different final concentrations of 0.25-64.00 mg/L.Microbroth dilution method was used to detect the MIC and MFC of above-mentioned mixture against C.albicans.The checkerboard method was used to detect in vitro inhibitory activity of AMPs-Mt6 combined with FLC,AmB,ITC,and 5-FC respectively against C.albicans,and the combined effect was determined by partial inhibitory concentration index(FIC Index).Results AMPs-Mt6 was an amphiphilic peptide with the positive charge number of 9,hydrophobicity of 0.451,α-helix ratio of 81%.The MIC of AMPs-Mt6 against C.albicans was 312.50 mg/L,and it had antibacterial effect on all detected bacteria.The MIC of AMPs-Mt6 against S.aureus,B.Subtilis,and P.aeruginosa was 625.00 m

关 键 词：白色念珠菌 家蝇抗真菌肽-1A 分子改造 抗菌肽-Mt6 抗菌活性 联合抗菌实验 

分 类 号：R379.4[医药卫生—病原生物学]