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

• 1:贵州医科大学基础医学院
• 2:贵州省普通高等学校病原生物学特色重点实验室

摘要(Abstract)：

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(156.25～2 500.00 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1 250.00 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 100 μL of bacteria suspension and AMPs-Mt6 dilution(156.25 to 2 500.00 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 mg/L; and MIC of AMPs-Mt6 against E. coli, S. paratyphi, and K. pneumonia was 1 250.00 mg/L. When AMPs-Mt6 combined with AmB, ITC, and 5-FC, FIC index value was 0.19, 0.36, and 0.37, respectively, showing synergistic effect; the FIC index value was 0.63 when AMPs-Mt6 combined with FLC, indicating additive effect. Conclusion Compared with template peptide MAF-1A, the physicochemical properties of AMPs-Mt6 are optimized. The antibacterial activity is enhanced and antibacterial spectrum is enlarged. The combination of AMPs-Mt6 with antifungal drugs(FLC, AmB, ITC, and 5-FC) can enhance the inhibitory effect of each drug against C. albicans.

关键词(KeyWords)： 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
Candida albicans;Musca domestica antifungal peptide-1A;molecular modification;antimicrobial peptides Mt6;antimicrobial activity;combined drug susceptibility test

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 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

DOI: 10.19367/j.cnki.2096-8388.2023.06.002

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