抗菌肽MAF-1A衍生物体外抗白念珠菌生物膜活性及机制研究

Effects and mechanisms of antimicrobial peptide MAF-1A derivatives anti-Candida albicans biofilm in vitro

目的探讨抗菌肽MAF-1A衍生物体外抗白念珠菌生物膜活性及潜在机制。方法采用微量稀释法检测MAF-1A衍生物对白念珠菌的MIC、MFC;通过扫描电镜等方法观察MAF-1A衍生物对白念珠菌生物膜形态学的影响;以XTT法测定MAF-1A衍生物对不同阶段生物膜活性的影响及生物膜80%抑制浓度(SMIC_(80));采用流式细胞术、激光共聚焦显微技术、qRT-PCR等分析MAF-1A衍生物抗白念珠菌生物膜的作用机制。结果MAF-1A衍生物对白念珠菌的MIC、MFC及SMIC_(80)均低于模板肽,分别为62.5μg/mL、125μg/mL和62.5~125μg/mL。MAF-1A衍生物可明显抑制白念珠菌的黏附和菌丝形成,且抑制作用呈剂量依赖性;可使菌细胞黏附及菌丝形成相关基因(ALS3、HWP1、SUN41、UME6)的mRNA表达量降低(t_(UME6)=12.42,P<0.001;t_(ALS3)=12.20,P<0.001;t_(SUN41)=7.206,P<0.001;t_(HWP1)=22.52,P<0.001);可使形成中的生物膜和成熟生物被膜活性明显降低(t_(250)=3.680,P<0.05;t_(500)=4.153,P<0.05;t_(1000)=4.934,P<0.05;t_(250)=0.5335,P<0.05;t_(500)=1.504,P<0.05;t_(1000)=6.431,P<0.05)。62.5μg/mL浓度的MAF-1A衍生物即可直接破坏白念珠菌成熟生物膜结构,引起成熟生物膜的细胞凋亡、ROS含量明显增加,线粒体膜电位显著降低。结论MAF-1A衍生物具有较好的体外抗白念珠菌生物膜活性,不仅能通过抑制细胞黏附、菌丝形成来干扰生物膜的早期形成,还能通过直接损伤以及ROS累积、线粒体膜电位去极化所引发的细胞凋亡来破坏成熟生物膜。

The purpose of this study was to investigate the effect and the underlying mechanisms of antimicrobial peptide MAF-1A derivatives against Candida albicans biofilms in vitro.In this research,MIC and MBC of MAF-1A derivatives against C.albicans was determined with microdilution method;the morphological changes of C.albicans biofilm were observed by scanning electron microscopy(SEM)and inverted microscope;the effects of MAF-1A derivatives on biofilm activity at different stages and 80%inhibitory concentration on biofilm(SMIC_(80))were determined by XTT assay.Flow cytometry,laser confocal microscope and qRT-PCR were used to investigate the mechanism for its anti-C.albicans biofilm activities.The MIC,MFC and SMIC_(80)of MAF-1A derivatives against C.albicans were lower than those of template peptides,which were 62.5μg/mL,125μg/mL and 62.5~125μg/mL,respectively.MAF-1A derivatives could significantly inhibit C.albicans adhesion and hyphal development in a dose-dependent manner.The expression of biofilm-related genes(ALS3,HWP1,SUN42,UME6)in C.albicans decreased after treatment with MAF-1A derivatives(t_(UME6)=12.42,P<0.001;t_(ALS3)=12.20,P<0.001;t_(SUN41)=7.206,P<0.001;t_(HWP1)=22.52,P<0.001).MAF-1A derivatives can significantly reduce the activity of both the forming and mature biofilms(t_(250)=3.680,P<0.05;t_(500)=4.153,P<0.05;t_(1000)=4.934,P<0.05;t_(250)=0.5335,P<0.05;t_(500)=1.504,P<0.05;t_(1000)=6.431,P<0.05).MAF-1A derivatives with a concentration of 62.5μg/mL could not only directly destroy the mature biofilm structure of C.albicans,but also significantly increase the cell apoptosis and ROS content,and significantly decrease the mitochondrial membrane potential.This study suggests that MAF-1A derivatives have remarkably inhibitory effects on C.albicans biofilm,the mechanisms may be associated with the inhibiting C.albicans adhesion and hyphal development,biofilm damage,promoting cell apoptosis.