核糖体蛋白RPS4A调控白念珠菌凋亡的机制研究

In recent years, more and more facts have proved that systemic fungal infection, in which Candida albicans is one of the main resources, is very hazardous to the increased patients with AIDS and tumor diseases. Induction of fungi apoptosis has been considered as a new antifungal strategy. We previously found that c. albicans ribosomal protein RPS4A could regulate cell apoptosis through CaMCA1. Also, there is no homologous protein of RPS4A in human cells, which indicates that RPS4A might be a new antifungal target. In this study, we are going to construct RPS4A-GFP fusion protein to identify the locus and expression of rps4ap during C. albicans apoptosis. Moreover，CaMCA1 will be deleted in rps4a mutant to investigate the role of CaMCA1 in the C. albicans apoptosis regulated by RPS4A. The impact of RPS4A on ribosome function and gene expression profiles will be determined. Furthermore, the method of yeast two-hybrid will be used to identify the interaction protein of RPS4A. The methods of Large DNA fragment deletion and site-directed mutagenesis will be used to study the structure-function relation of RPS4A. Through this study, the molecular mechanisms of RPS4A in regulating C. albicans apoptosis will be clarified and RPS4A will be proved as new antifungal drug target.

近年来，以白念珠菌为主的深部真菌感染日益普遍，对艾滋病，肿瘤等病人构成严重危害，诱导细胞凋亡是一种抗真菌感染新途径。我们在前期研究中发现白念珠菌核糖体蛋白RPS4A可通过影响凋亡因子CaMCA1功能调控细胞凋亡,且该蛋白与人细胞蛋白无同源性，提示RPS4A是潜在的抗真菌药物新靶标。在本项目中，我们拟构建RPS4A-GFP融合蛋白表达菌，以明确RPS4A在白念珠菌细胞凋亡过程中的时空秩序。此外，在已构建的RPS4A基因缺失菌中进一步敲除CaMCA1基因，考察CaMCA1在RPS4A调控白念珠菌凋亡中的作用。同时，考察RPS4A对白念珠菌核糖体功能及基因表达谱的影响。进一步地，采用酵母双杂交技术寻找与RPS4A相互作用蛋白，采用基因大片段缺失及定点突变技术研究其结构功能关系，以阐明RPS4A调控白念珠菌细胞凋亡的分子机制及其靶标作用，为抗真菌感染提供新思路。

近年来，以白念珠菌为主的深部真菌感染日益普遍，对艾滋病及肿瘤等病人构成严重危害，诱导细胞凋亡是一种抗真菌感染新途径。本项目基于我们在前期研究中发现的白念珠菌核糖体蛋白RPS4A可调控细胞凋亡,对RPS4A的基因功能作进一步深入研究。生物信息学分析显示，白念珠菌Rps4p由同源基因RPS4A和RPS4B编码，两者所编码的蛋白仅有一个氨基酸的差异。我们用“Ura-Blaster”基因敲除策略在CAI4菌中分别构建了rps4aΔ 和 rps4bΔ基因缺失菌。研究表明，基因RPS4A在白念珠菌的生命活动中比基因RPS4B发挥更加重要的作用，基因RPS4B基本上处于沉默状态。实验表明，RPS4A基因的缺失对白念珠菌核糖体功能无明显影响，但RPS4A基因缺失菌对凋亡诱导剂过氧化氢和两性霉素B更敏感度。另外，与野生型菌相比，RPS4A基因缺失菌表现为生长速率降低、菌丝形成能力下降、生物被膜形成能力减弱、毒力降低、侵袭力也相应的减弱。基因表达谱研究表明，白念珠菌缺失RPS4A基因后导致390个基因发生表达水平发生变化，其中198个基因表达上调，192个基因的表达下调，差异表达基因功能主要涉及能量代谢、物质转运及氨基酸合成等。本项目研究未抗真菌感染提供了新思路。