HtrA对白色念珠菌致病性的影响及作用机制研究

C.albicans is an important opportunistic fungi in clinical infections. The virulence factors and mechanisms were not elucidated until now. Our group finished the first genome of C.albicans （CA6184） in this district. Subsequently, we found High Temperature Requirement protein A of C.albicans (CaHtrA) was different from other species, which has only one copy in the genome, and two DegQ domain. Other pathogens have two or more copies, and one DegP/Q domain. In addition, it was reported that this protein was related to the pathogenicity of C.albicans. It points out CaHtrA might be an important virulence factor. Therefore, we firstly acquire the recombinant protein and its antibody, then confirm the protease activity and subcellular localization of CaHtrA. It can provide more information for our forward researches. In addition, we plan to construct the mutant strain of CaHtrA. After that, we would like to compare the pathogenicity of CaHtrA-/- and wild type in animal model. Furthermore, compared proteome will be carried out to study the proteins could be regulated by CaHtrA. Finally, we will confirm the proteomic results to elucidate the protein-protein interaction and pathway related to CaHtrA. Our study could understand the function of fungal HtrA. It could elucidate the pathogenicity, related mechanism of CaHtrA, and provide more information for studying Candidasis in future.

白色念珠菌是临床上一类重要的条件致病性真菌，其致病因子及致病机制尚未完全阐明。本课题组去年完成国内首个白色念珠菌（CA6184）全基因组测序后，进一步发现，HtrA在白色念珠菌中为单拷贝双DegQ结构域，不同于其他病原菌HtrA的多拷贝单DegP/Q结构域。据文献报道，HtrA与白色念珠菌的致病可能有一定联系，但尚未得到证实。在此基础上，本项目拟首先获得该基因的重组蛋白及多克隆抗体，体外验证HtrA是否具有HtrA的典型功能，并确定亚细胞定位，从而了解该蛋白可能的作用机制；其次，构建基因缺失菌株，利用动物模型，比较突变株与野生株的差异，确证HtrA的致病性；同时，利用定量蛋白质组技术，比较突变株与野生株蛋白质组的差异，阐明HtrA发生相互作用的蛋白，及影响的信号转导通路和重要毒力基因。本研究为了解真菌HtrA的功能、阐明其在白色念珠菌中的致病功能及作用机制提供重要的依据。

白色念珠菌是一类重要的人类条件致病性真菌。该类真菌的关键致病因子尚未完全阐明。本课题组前期发现白色念珠菌基因组中存在的HtrA蛋白具有独特的功能，因此，本研究针对白色念珠菌的HtrA蛋白进行系统性研究。本课题的研究发现：1.白色念珠菌HtrA蛋白定位于细胞质、靠近细胞膜一侧，在菌丝相这一特征尤为明显；2.白色念珠菌HtrA蛋白的219和220位丝氨酸是其关键的酶活性位点，与该蛋白发挥丝氨酸蛋白酶作用直接相关；3.白色念珠菌HtrA蛋白在其生物膜形成过程中发挥重要作用，并与该类真菌的致病性密切相关；4.在白色念珠菌HtrA蛋白影响生物膜形成的机制中，我们发现该蛋白受到上游转录因子Bcr-1的调控，并且该蛋白可能直接调控生物膜形成的关键因子Als1、Als3等。综上所述，定位于白色念珠菌细胞质膜一侧的HtrA蛋白，受到转录因子Bcr-1的调控，并影响生物膜形成关键蛋白Als1、Als3，从而影响白色念珠菌的生物膜形成及其致病力；即白色念珠菌HtrA蛋白可能是其关键致病因子之一。