基于细胞凋亡的植物防御素WsAFP1抗真菌机制研究

Candida albicans(C. albicans) is currently one of the most common pathogens of systemic fungal infection. With the long term and excessive use of the antifungal drugs that are most commonly used in the clinical treatment of systemic fungal infections, drug-resistant strains were constantly produced and led to a growing number of treatment failure. Therefore, developing the new antifungal drug and looking for new mechanisms of antifungal or antifungal drug targets are becoming a hot topic in antifungal drug research and development.Plant defensins, small basic peptides that are inhibitory against a range of plant and human pathogens, are the important role in the innate immune system of plants.The plant defensins are hopefully developed an new antifungal drug owing for their excellent characteristics such as safety, small side effects and minor possibility for generating drug resistant strains. In this project, we will focus here on studies aimed at unraveling the antifungal mechanism of a novel plant defensin WsAFP1 against C. albicans based on fungi apoptosis. We plan to determine the c. albicans membrane lipid that interact with WsAFP1 using sphingolipid key gene mutations strains and inhibitors for key enzyme of complex lipid synthetic pathway. Then we will investigate the effects on permeability of cell membrane and influence of cell growth after WsAFP1 interaction with c. albicans membrane lipid.We will also measure the change of reactive oxygen species (ROS) level in cell of c. albicans and further research on the induction of apoptosis pathway in c. albicans. At the same time, we will try to explore intracellular receptor in c. albicans for WsAFP1 using laser confocal microscopy and tandem affinity purification technology.In the end, we will elucidate the mechanism that WsAFP1 inhibit the growth of c. albicans based on cell apoptosis mechanism, try to discover new antifungal drug targets and build the foundation for development of a new antifungal drug.

深部真菌特别是白念珠菌感染发生率有逐年上升的趋势，而对临床上使用较广的抗真菌药物耐药菌株也越来越多，因此开发新的抗真菌药物或发现新的抗真菌药物靶点成为抗真菌药物研发的热点之一。植物防御素是植物组织器官中广泛存在的广谱抗真菌肽，有望发展为一种新的安全抗真菌药物替代品。本项目以我们新发现的植物防御素WsAFP1和白念珠菌为材料，利用鞘脂类合成通路关键基因突变菌株或鞘脂、甾醇等合成途径关键酶抑制剂确定WsAFP1在白念珠菌膜上相互作用的脂类，及其对白念珠菌细胞膜通透性和细胞生长的影响；研究WsAFP1与白念珠菌相互作用后对白念珠菌胞内活性氧水平的影响及其原因，进一步研究其诱导白念珠菌细胞凋亡途径及机制；同时利用激光共聚焦显微镜和串联亲和纯化技术探索WsAFP1在白念珠菌胞内受体。阐明WsAFP1诱导白念珠菌细胞凋亡及抑制其生长的机制，为发现新的抗真菌药物靶点，开发新的抗真菌药物打下理论基础。

开发新的抗真菌药物或发现新的抗真菌药物靶点成为抗真菌药物研发的热点之一。植物防御素是植物在长期进化过程中为了抵抗外界病原微生物或不良环境的侵害而形成的一种天然防御物质，具有多种功能，如抑制真菌、细菌生长和肿瘤细胞增殖等。本项目以新发现的植物防御素WsAFP1为材料，确定了WsAFP1在白念珠菌细胞膜上相互作用的鞘脂为葡萄糖神经酰胺（GlcCer）；测定了rWsAFP1对白念珠菌孢子萌发的抑制作用；测定了rWsAFP1对白念珠菌胞内ROS水平的影响；测定 rWsAFP1 和白念珠菌菌体总蛋白之间的相互作用的参数；解析了rWsAFP1的蛋白质晶体结构，为发现新的抗真菌药物靶点，开发新的抗真菌药物打下理论基础。