Functional characterisation of Ago2 as erythrocyte host protein that is translocated to the Plasmodium parasite.

疟疾是由真核单细胞疟原虫感染红细胞引起的，是最致命的人类传染病之一。现有的大多数抗疟疾药物靶向寄生虫蛋白，这些蛋白可迅速突变，使寄生虫产生耐药性。鉴于这种对常规抗疟疾药物的耐药性迅速蔓延，现在最重要的是确定新的药物靶点，特别是不易发生耐药性突变的基本宿主蛋白。一个候选靶点是宿主蛋白Argonaute 2 (Ago2)，它在红细胞中表达。有趣的是，最近的研究发现，Ago2易位到啮齿动物和人类疟原虫的血分期，并且在啮齿动物疟原虫中人工过表达Ago2特异性地影响了雌性配子体中mRNA的储存。这些观察结果提出了我的假设，即Ago2在寄生虫中发挥了未知但可能必不可少的功能。本文的目的是全面剖析Ago2在疟原虫血期生物学中的作用。我将研究人类和啮齿动物的疟疾寄生虫，有以下三个具体目标：(1)利用超分辨率显微镜和电子显微镜确定Ago2的精确亚细胞定位。(2)疟原虫在Ago2缺陷红细胞中的表型分析，我将通过化学试剂抑制Ago2或通过基因工程红细胞中Ago2蛋白的耗尽来产生Ago2缺陷红细胞。(3) Ago2 RNA和蛋白质相互作用伙伴的功能特征，将通过Ago2的免疫沉淀、RNA测序和质谱分析来鉴定。总之，该项目将首次阐明宿主蛋白转运到疟原虫血液阶段的功能。这项研究不仅将为宿主定向治疗疟原虫的潜在靶点提供有价值的见解，而且还将获得新的科学技能，这将有助于我未来的学术生涯。

Malaria, caused by eukaryotic unicellular Plasmodium parasites infecting erythrocytes, is one of the deadliest human infectious diseases. Most antimalarial drugs available target parasite proteins which can rapidly mutate, rendering parasites resistent. Given the rapidly increasing spread of such resistances to conventional anti-malarial drugs, it is now of utmost importance to identify novel drug targets, in particular essential host proteins which are less prone to resistance mutations. One candidate target is the host protein Argonaute 2 (Ago2), which is expressed in erythrocytes. Intriguingly, recent studies have found that Ago2 translocates into rodent and human Plasmodium blood stages, and that the artificial overexpression of Ago2 in rodent Plasmodium specifically affects mRNA storage in female gametocytes. These observations give rise to my hypothesis that Ago2 fulfills yet unknown but potentially essential functions in the parasite. The aim of this proposal is the comprehensive dissection of the role of Ago2 in the blood stage biology of Plasmodium. I will study both human and rodent malaria parasites with the following three specific aims: (1) Determination of the precise subcellular localisation of Ago2 using super resolution microscopy and electron microscopy. (2) Phenotyping Plasmodium in Ago2-deficient erythrocytes, which I will generate either by inhibition of Ago2 using chemical agents, or by depletion of Ago2 protein in genetically engineered erythrocytes. (3) Functional characterisation of Ago2 RNA and protein interaction partners that will be identified by immunoprecipitation of Ago2 followed by RNA sequencing and mass spectrometry. In summary, this project will for the first time elucidate the function of a host protein that is translocated into the Plasmodium blood stage. This research will not only provide valuable insight into a potential target for host-directed therapy of Plasmodium, but I will also acquire novel scientific skills that will foster my future academic career.