Phb-2在无绿藻病发病机制及宿主固有免疫中的作用研究

Prototheca is an opportunistic pathogen which can cause cutaneous and systemic infections. Increasing morbidity and mortality of protothecosis have draw much attention. So far no research on the pathogenesis of protothecosis has been reported. In recent research, it is presumed that virulence factors related with proliferation and oxidative stress are critical in prototheca pathogenicity. Our group has carried out relatively consummate research on the morphology, molecular biologic identification, virulence evaluation and systemic infection animal models of this pathogen, demonstrating that different prototheca strains differ in pathogenicity. iTRAQ, one of the latest technique of proteomics, was used for our further research. We found that the content of phb-2 was significantly higer in the pathogenic strains than the non-pathogenic ones. Besides, by western blot method,we detected that on oxidative stress occasion phb-2 was compensatory increased and transfered to mitochondria in the prototheca cell. On the basis of our previous results and the developments in the study of phb-2, our project will knock out the coding genes of phb-2 in the pathogenic strains of prototheca and carry out related genic functional study of phb-2 subsequently, which includes analysis of the growth of colony, animal virulence and effect on macrophage phagocytosis. The project is to explore the effect of phb-2 on the pathogenesis of prototheca, and further to provide theoretical foundation for researches of novel antimycotic drug targets.

无绿藻是一种条件致病真菌，其引起皮肤及系统感染的报道逐年增多，死亡率高，对免疫受累人群危害尤大，已引起国内外较多关注。目前尚无对其致病机制研究的报道。本课题组前期对无绿藻分子生物学鉴定、毒力鉴定及感染动物模型进行了较系统的研究，发现不同菌株致病性存在差异。通过蛋白质组学iTRAQ技术检测到prohibitin-2(phb-2)含量在致病性菌株中较非致病性菌株显著升高，并进一步通过蛋白质印迹试验检测发现，氧化应激无绿藻细胞中phb-2表达增加并向线粒体移位，因此phb-2可能是与无绿藻致病性相关的重要毒力因子。本课题拟比较phb-2编码基因缺失前后菌株的生长情况差别；构建小鼠无绿藻感染模型，研究phb-2缺失前后菌株致病力的差异；检测巨噬细胞对phb-2缺失前后菌株的吞噬杀伤作用差异，探索phb-2对宿主固有免疫的影响。通过以上研究了解无绿藻的致病机制，为研究抗真菌药物新靶点提供理论基础。

无绿藻是一种罕见的条件致病真菌，可引起人与动物的感染，临床诊断比较困难，目前主要依靠微生物实验室进行诊断和鉴定。无绿藻导致的深部感染在我国极为罕见，迄今我国大陆地区虽有7例皮肤型无绿藻病的报道，但尚无系统性无绿藻病的报道。 .课题组首次绘制出了人类感染的中型无绿藻致病株的基因组精细图，通过蛋白质组学和转录组学发现了部分中型无绿藻致病株与环境株间的差异基因，这些基因差异可能通过提高中型无绿藻致病株在缺乏葡萄糖环境中的生存能力，以及对巨噬细胞产生的抗性造成致病性，研究鉴定得到的差异表达基因为研究无绿藻潜在毒力因子提供了理论基础。同时建立并优化了无绿藻的基因敲除方式，通过RNAi对目标基因进行筛选，我们已成功构建数个上述基因表达下调的无绿藻致病株。这些结果初步探索了中型无绿藻致病株可能的致病机制，并为今后的研究奠定了基础。.我们还通过无绿藻致病菌株与非致病的环境菌株与巨噬细胞体外共培养模型的构建，发现不同的菌株吞噬感染巨噬细胞一定时间后存活率存在差异，iNOS mRNA表达和蛋白表达水平存在也有显着差异，可以部分解释造成菌株间致病性的不同，以及解释无绿藻感染的方式，为后续对无绿藻致病方式更深入的探索研究提供了思路。