人工合成家蝇抗真菌肽MAF-1A体外抗真菌效果及扫描电镜观察

Antifungal Effect and Scanning Electron Microscopy for the Synthetic Musca domestica Antifungal Peptide-1A( MAF-1A)

目的探讨人工合成家蝇抗真菌肽MAF-1A结构特点及其抗真菌效果。方法运用生物信息学软件分析人工合成MAF-1A的结构特征;采用液体微量稀释法、MTT法和沙氏琼脂平板培养计数法分别检测MAF-1A对白色念珠菌、光滑念珠菌、热带念珠菌、克柔念珠菌、近平滑念珠菌和新生隐球菌的抗菌活性及最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并绘制时间-杀菌曲线;采用扫描电镜观察在MIC浓度下对白色念珠菌超微结构的影响。结果生物信息学软件分析结果显示MAF-1A为一阳离子多肽,分子量3022.5Da,pI值9.23,碱性氨基酸含量26.9%,带2个正电荷,结构较为稳定,脂肪指数71.54,其总平均疏水性数-1.081,预测为一非跨膜多肽,二级结构以α螺旋结构为主的线状肽结构。MAF-1A对白色念珠菌敏感株及耐药株均显示出抗菌活性,对检测酵母菌的MIC值在0.1~0.8mg/ml、MBC值在0.2~0.9mg/ml;杀菌曲线显示,MIC浓度MAF-1A作用真菌2h至14h逐渐发挥杀菌活性。扫描电镜显示MAF-1A作用白色念珠菌后形态发生改变,菌体表面粗糙,可见细胞穿孔样变,严重病变细胞表面呈碎片状,刺状突起,胞质流失至细胞皱缩死亡。结论 MAF-1A为阳离子α-螺旋结构线性多肽,对常见的致病性念珠菌包括耐药性菌株具有抗菌活性,可通过破坏真菌细胞表面结构发挥杀菌作用。

Objective To investigate the antifungal activity and structure feature of the synthetic Musca domestica antifungal pep- tide - 1A（ MAF - 1A）. Methods The structure feature of the synthetic peptide MAF - 1A was analyzed by the bioinfol^natics soft- ware and the antifungal activity, minimal inhibition concentration（ MIC ） and minimal bactericidal concentration （MBC） of MAF - 1A against C. albicans , C. glabrata , C. krusei , C. tropicalis , C. parapsilosis and C. neoformans were determined by the broth microdi- lution testing and MTT assay. The time - killing curves had been plotted with the colony counts and uhrastructural observation of C. albicans exposing to the MIC concentration of MAF - IA was also performed by Scanning Electron Microscopy method. Results The theoreticalmnlecular weight of MAF - 1A was 3022.5Da and the isoeleetric point was 9.23. MAF - 1A had two positive char- ges and basic amino acid content of was 26.9 percent. The MAF - 1A structure was stable and the aliphatic index was 71.54, the grand average of hydrophobicity（GRAVY） was - 1. 081. It predicted that MAF - 1A was a linear cationic polypeptide with abun- dant α -helix. MAF - 1A showed an antifungal activity against yeast cells including the resistant strains. The MICs and MBCs were 0.1 -0. gmg/ml and 0.2 ~ 0.9mg/ml respectively. MAF - 1A showed the strongest antifungal activity in the early 14h, and then weakened gradually. Compared with the MAF - 1A - free controls, the cell surface appeared the pathological changes in MAF - 1A group. Conclusion MAF - 1A was a linear cationic polypeptide with abundant α- helix and had an antifungal activity a- gainst the common pathogenic Candia species through inference on the cell surface structure.