新型抗菌肽Mt-22S3对白念珠菌细胞壁及细胞膜的作用  

Effects of the novel antimicrobial peptides Mt-22S3 on cell wall and cell membrane of Candida albicans

作　　者：李彩多 曾晔 石艳萍 张迎春 吴建伟 陈峥宏 王涛 LI Caiduo;ZENG Ye;SHI Yanping;ZHANG Yingchun;WU Jianwei;CHEN Zhenghong;WANG Tao(School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Microbiology and Parasitology of Institution of Higher Learning 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11期1329-1336,共8页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目(黔科合支撑〔2020〕4Y236);贵州省教育厅滚动支持省属高校科研平台项目(黔教技〔2022〕019)。

摘　　要：目的探讨家蝇抗真菌肽-1A(MAF-1A)突变体-22S3(Mt-22S3)对白念珠菌(C.albicans)细胞壁及细胞膜的作用。方法取对数生长期C.albicans菌落配制菌悬液,与31.3~500.0 mg/L Mt-22S3进行孵育,采用微量肉汤稀释法测定Mt-22S3抗C.albicans最低抑菌浓度(MIC)和最低杀菌浓度(MFC);取对数生长期C.albicans菌悬液,分别与0、125、250及500 mg/L Mt-22S3作用12 h,采用电镜观察Mt-22S3对C.albicans细胞壁、细胞膜的影响;使用荧光显微法观察Mt-22S3对C.albicans细胞膜通透性的影响;以0、250 mg/L Mt-22S3分别作用C.albicans细胞24 h,采用实时荧光定量PCR(qRT-PCR)法检测Mt-22S3对C.albicans细胞壁合成相关基因[几丁质合成酶基因1(CHS1)、几丁质合成酶基因2(CHS2)、几丁质合成酶基因3(CHS3)、β-葡聚糖合成相关蛋白kre1基因(KRE1)、β-葡聚糖合成相关蛋白kre6基因(KRE6)及甘露糖蛋白65基因(MP65)]和细胞膜麦角甾醇合成相关基因[角鲨烯环氧化酶基因(ERG1)、C-22甾醇去饱和酶基因(ERG5)、甾醇24-C-甲基转移酶基因(ERG6)及5-甲基四氢蝶酰基三谷氨酸-高半胱氨酸甲基转移酶基因(MET6)]信使RNA(mRNA)的表达。结果Mt-22S3对C.albicans的MIC为125 mg/L,MFC为250 mg/L;经Mt-22S3作用后,扫描电镜可见C.albicans菌细胞出现形态不规则、表面褶皱等细胞病理改变,透射电镜显示C.albicans细胞壁完整性未见明显改变,但细胞膜与细胞壁分离,细胞膜出现松弛、不连续及产生孔洞等病理改变;C.albicans细胞膜的通透性随着Mt-22S3浓度升高而增高,且呈浓度依赖性;与0 mg/L Mt-22S3组相比,经Mt-22S3作用的C.albicans细胞壁CHS1、CHS2、CHS3、KRE1、KRE6及MP65 mRNA表达比较差异无统计学意义(P>0.05),细胞膜ERG1、ERG5、ERG6及MET6 mRNA表达下降(P<0.0001)。结论Mt-22S3不仅可直接破坏C.albicans细胞膜结构,使细胞膜的通透性增高,还能降低细胞膜的稳定性。Objective To investigate the effects of mutant-22S3 of Musca domestica antifungal peptide-1A(Mt-22S3)on the cell wall and membrane of Candida albicans(C.albicans).Methods C.albicans colonies in logarithmic growth phase were used to prepare bacterial suspension and was incubated with Mt-22S3(31.3-500.0 mg/L).The minimum inhibitory concentration(MIC)and minimum fungicidal concentration(MFC)against C.albicans were examined by microbroth dilution method.The suspensions of C.albicans at logarithmic growth stage were treated with 0,125,250,and 500 mg/L Mt-22S3 for 12 h.The effects of Mt-22S3 on the cell wall and cell membrane of C.albicans were observed by electron microscopy.The effect of Mt-22S3 on cell membrane permeability of C.albicans was observed by fluorescence microscopy.C.albicans cells were treated with 0 and 250 mg/L Mt-22S3 for 24 h.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the effects of Mt-22S3 on the messenger RNA(mRNA)expression levels of C.albicans cell wall chitin synthesis gene 1(CHS1),chitin synthase gene 2(CHS2),chitin synthase gene 3(CHS3),beta-glucan synthesis-associated protein kre1 gene(KRE1),beta-glucan synthesis-associated protein kre6 gene(KRE6),65-kD mannoprotein gene(MP65),squalene monooxygenase gene(ERG1),C-22 sterol desaturase gene(ERG5),sterol 24-C-methyltransferase gene(ERG6),and 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase gene(MET6).Results The MIC and MFC of Mt-22S3 to C.albicans were 125 mg/L and 250 mg/L.After treatment with Mt-22S3,scanning electron microscopy revealed that C.albicans cells showed irregular morphology and surface wrinkles,and transmission electron microscopy showed that the integrity of C.albicans cell wall was not significantly changed,but the cell membrane was kept separate from the cell wall,resulting in pathological changes such as relaxation,discontinuity,and formation of pores in the cell membrane.The permeability of C.albicans cell membrane increased with the increase of Mt-22S3 concentration in a concent

关 键 词：白色念珠菌 细胞壁 细胞膜 通透性 抗菌肽 稳定性 麦角甾醇 

分 类 号：R978.5[医药卫生—药品]