利用线虫模型发现白念珠菌关键致病因子及抗真菌活性化合物的研究

侵袭性真菌感染已成为免疫功能低下人群和重症监护病人死亡的重要原因，其中白念珠菌感染最为常见，研究关键致病因子及抗真菌新药尤为重要。我们从哈佛医学院引进了高通量低成本的线虫模型，将线虫作为感染宿主，对真菌遗传学储备中心4个白念珠菌基因突变菌库680株突变菌的致病力进行了筛选评价，发现了11个可能的关键致病因子，其中6个属于激酶类。本项目计划①用先进的SAT1 Flipper法分别完成上述11个基因敲除菌和回复菌的构建，用线虫模型和小鼠模型评价各敲除菌、回复菌及亲本菌的致病力，明确关键致病因子；②经生物信息学分析确定可作为药靶的关键致病因子，模建蛋白结构，虚拟筛选化合物样品库中20862个化合物；③用线虫模型评价通过虚拟筛选的化合物和激酶抑制剂的抗真菌活性及毒性；④用小鼠模型对化合物进行再评价，进而发现高效低毒的抗真菌活性化合物。本项目可以为抗真菌药靶的发现和抗真菌新药的研发奠定工作基础。

Invasive fungal infection has become an important cause of death in immunocompromised population and intensive care patients, of which Candida albicans infection is the most common. Currently, our knowledge on Candida virulence factors is limited, and clinical antifungal drugs are inadequate. It is of great importance to discover the key virulence factors of Candida and to develop some antifungal new drugs. Here, we introduced a high-throughput low-cost nematode model from Harvard Medical School. Using Caenorhabditis elegans as the host of infection, we screened 680 mutants from 4 mutant libraries of Fungal Genetics Stock Center and found 11 C. albicans mutants with significantly decreased virulence. Of the 11 mutant genes, 6 encode kinases. This project is designed to reveal the key virulence factors of C. albicans and to discover some new antifungal compounds: ① Developing gene knockout strains and reconstitute strains of the 11 genes using an advanced SAT1 Flipper strategy; evaluating the virulence of the gene knockout strains, the gene reconstitute strains and the parental strain using both C. elegans model and mouse model to identify the key virulence factors. ② Determining several key virulence factors as the potential drug targets based on those work above and with the aid of bioinformatics analysis, literature review and studies on upstream and downstream pathways; presenting the structure of the target proteins using our computer-aided platform; virtual screening 20862 compounds from our compound library. ③ Evaluating the antifungal activity and toxicity of the kinase inhibitors and of the compounds which have passed the virtual screening using the C. elegans model. ④ Re-evaluating the active compounds to confirm those with high efficiency and low toxicity using the mouse model. This project is meaningful for antifungal target discovery and antifungal new drug development.