基于化学蛋白组学技术研究青蒿素通过作用于宿主红细胞膜的抗疟机理

Malaria is a life-threatening disease caused by the protozoan parasite Plasmodium in the world. Artemisinin-based combination therapies (ACTs) has been adopted as a first-line treatment for uncomplicated Plasmodium falciparum malaria by World Health Organization (WHO). However, resistance to artemisinins and partner drugs is now causing the failure of P. falciparum ACTs in southeast Asia. Therefore, it is critical to understand artemisinin's mechanism of action in order to find an effective method to conquer the resistance of malaria parasites in clinical or to design artemisinin derivatives with better antimalarial activity. our previous work confirmed that the total protein expression of Plasmodium falciparum 3D7-infected red blood cell (IRBC) membrane was down-regulated after 4 h treatment using 500 nM dihydroartemisinin. We thus generate the hypothesis that artemisinin exerts antimalarial effects by directly acting on the plasma membrane of IRBC. This project intends to first use an alkyne-tagged artemisinin analogue coupled with biotin (AP1) to identify artemisinin covalent binding protein targets on IRBC membrane based on chemical proteomics in the P. falciparum 3D7 and P. berghei ANKA (PbA)-infected C57BL/6 (B6) mouse models. Then using pull-downs followed by immunoblotting to validate some selected artemisinin targets and also using heterologous expression system to validate the targets’interaction with AP1 in vitro, selecting a specific protein for artemisinin's action. Finally, we attempt to further explore the function of the target protein in the anti-malarial process of artemisinin by using several gene manipulation methods related to gene silencing and overexpression, to reveal the antimalarial mechanism of artemisinin. Our study will provide a critical guideline for artemisinin resistance control in malaria parasites and lay the foundation for developing new artemisinin-based antimalarial drugs.

疟原虫引起的疟疾是一种威胁人类生命安全的世界性严重疾病。青蒿素联合疗法是治疗疟疾的一线药物。然而疟原虫的青蒿素耐药现象日渐严重，因此，揭示青蒿素抗疟机制以寻找干预疟原虫耐药的方法已经成为临床中亟待解决的实际问题。本课题组前期发现双氢青蒿素治疗恶性疟原虫3D7后，3D7感染红细胞（IRBC）质膜总蛋白表达量下调。因此，我们建立了青蒿素通过直接作用IRBC质膜发挥抗疟作用的假说。本项目拟首先利用青蒿素活性探针（AP1），以3D7和鼠伯氏疟原虫（PbA）为模型，运用化学蛋白组学方法，获得青蒿素在IRBC膜上的关键靶蛋白；进一步利用Western blot验证靶蛋白的表达、利用异源表达系统验证其与AP1的结合，筛选出最特异结合蛋白；最后试图克隆该基因，构建干涉载体和过表达载体，探索特异靶蛋白的功能，揭示青蒿素抗疟机制，为控制青蒿素抗药性提供指导，为研发新型抗疟药奠定基础。

青蒿素联合疗法是WHO认定的国际一线抗疟药，但青蒿素的作用机制尚不清楚。本研究通过联合磁珠分离法和山梨醇同步法，构建了一种能够快速获得恶性疟原虫不同生长周期的同步化方法，可短时间内获得大量窗口期较窄的疟原虫。根据研究计划，本研究运用化学蛋白组学，利用青蒿素探针和生物素-链霉亲和素系统富集纯化蛋白质，鉴定出青蒿素可能作用的五个蛋白靶点为pfEMP1，pfHly III，pfSURFIN，pfROPE及TRAP like protein。已用crispr/cas9技术构建靶点蛋白的GFP载体，与青蒿素荧光探针进行共定位分析，其功能验证实验进一步进行中。已知疟原虫的寄生可诱导感染红细胞(iRBC)膜的形态变化，本研究通过光学显微镜和扫描电子显微镜观察发现DHA可引起iRBC膜的形态变化。为了了解青蒿素是否与iRBC膜蛋白相互作用，我们进一步利用RNA-seq分析了双氢青蒿素(DHA)对疟原虫基因转录水平的影响，发现rhoptry，myosin complex，transporter等与疟原虫蛋白的转运、抗原变异及其免疫逃逸等过程相关的基因变化显著；利用定量蛋白组学分析发现感染红细胞膜蛋白表达谱的变化主要集中于结构蛋白活性及蛋白质输出等关键生物过程。进一步发现DHA治疗降低了var基因PF3D7_0415700的表达和PF3D7_0900100，且呈剂量依赖性。蛋白质印迹和免疫荧光分析表明DHA处理下调了var基因编码蛋白红细胞膜蛋白-1(pfEMP1)。pfEMP1敲除显著增加了青蒿素敏感性。结果表明pfEMP1可能参与了DHA抗疟过程，并可成为一个潜在的抗疟治疗新靶点，本研究为进行青蒿素抗疟机制研究提供了研究方向，为进一步开发新的抗疟药物提供了理论基础。