疟原虫子孢子募集H因子抑制补体杀伤的成分鉴定及功能研究

Complement is the first line of host defense against a variety of infection, but its role in the malaria liver-stage development is still unknown. Our preliminary studies showed that infection of sporozoites could significantly activate complement, but it has no effect on sporozoites. Our further study showed that the complement inhibitory protein factor H could bind to the surface of sporozoites, and neutralization of factor H significantly inhibited the developmental of sporozoites in hepatocytes, indicating that Plasmodium sporozoites could recruit factor H to evade complement-mediated killing. However, the underlying mechanism is unknown. In this proposal, therefore, we aim to exclude the role of other regulatory factors such as CD59 contributing to sporozoites evasion of complement-mediated killing firstly. Next, we intend to screen and identify the sporozoites component that could be able to bind with factor H by co-immunoprecipitation and pull-down assay, and investigate its role in sporozoites evasion of complement-mediated killing. Additionally, the functional domains of the sporozoites component binding with factor H would be predicted and identified by bioinformatics and protein truncation experiments. This work is helpful for uncovering a novel approach utilized by Plasmodium sporozoites to escape host immunity, and providing an important theoretical foundation for the design of preventive drugs based on the new immune evasion mechanism.

补体是机体抗感染的第一道防线，但是其在肝期疟原虫发育中的作用并不清楚。课题组前期研究发现，虽然子孢子感染能活化补体，但是体内外实验证实活化的补体并不能杀伤子孢子。进一步研究发现，补体调节蛋白H因子能结合到子孢子表面，而中和H因子可显著抑制子孢子在肝细胞内的发育，提示疟原虫子孢子能募集H因子逃避补体杀伤，但具体分子机制并不清楚。鉴此，本项目拟在前期研究基础上，首先排除其他调节因子如CD59在子孢子抑制补体对其杀伤中的作用，然后，采用免疫共沉淀、pull-down技术筛选和鉴定与H因子结合的子孢子成分，并验证其在子孢子抑制补体杀伤中的作用。最后，通过生物信息学和蛋白截短实验预测并鉴定与H因子结合的子孢子成分的功能域。这不但可以揭示疟原虫子孢子逃避机体免疫的新机制，而且可为基于该逃避机制设计预防性药物提供重要理论依据。

补体是机体抗感染的第一道防线，但是其在肝期疟原虫发育中的作用并不清楚。本项目研究发现子孢子感染能活化补体C3和C5，但活化的C3不参与对子孢子的杀伤，是因为子孢子募集H因子从而抑制了补体的杀伤。现已筛选出2个与H因子结合的疟原虫子孢子候选成分，正在进一步确认并鉴定功能域，以及验证其在子孢子抑制补体杀伤中的作用。此外，我们还发现子孢子活化的补体C5a/C5aR信号会诱导宿主表达一种新型免疫负性调控分子纤维蛋白原样蛋白2（FGL2），后者通过抑制肝脏促炎因子IFN-γ表达，进一步促进肝期疟原虫发育。进一步地，我们还在红内期感染中研究了FGL2的作用，发现红内期疟原虫感染也可活化FGL2，后者通过抑制宿主固有免疫促进疟原虫增殖(science advances, 2020)。最后，我们对本研究获得的重要目标分子进行了潜在应用研究，并申请了3项国家发明专利。这些研究提示疟原虫可以巧妙的利用活化的补体来上调FGL2表达，从而抑制宿主的免疫应答逃避杀伤，促进其自身发育。本项目不仅揭示了红外期疟原虫新的免疫逃避机制，还可为设计疟疾防控策略提供新思路和新靶点。