Molecular basis of effector protein export in the malaria parasite Plasmodium falciparum

项目摘要 疟疾是一种毁灭性的寄生虫病，每年影响2亿多人，导致近 每年有50万人死亡。截至2018年，世界卫生组织估计，大约有38亿人 世界上一半的人口面临感染疟疾的风险，抗药性寄生虫的崛起创造了 迫切需要新的抗疟疾药物。虽然大多数细胞内病原体只输出有限的 效应器蛋白与现有的宿主细胞代谢机制--导致疟疾的寄生虫疟原虫--的协同作用 在血液阶段，恶性疟原虫将超过10%的蛋白质组输出到宿主人类红细胞中。 它的生命周期。恶性疟原虫出口组中的数百种蛋白质广泛地重塑宿主红细胞， 建立输入营养物质、输出废物和逃避宿主免疫系统所需的基础设施。这个 这数百种蛋白质的出口因为疟疾寄生虫隐藏在一个 寄生空泡是在入侵过程中寄主细胞质膜内陷形成的空泡。 在分泌到PV后，要出口的蛋白质必须打开并通过PV运输 在依赖于ATP的过程中，膜(PVM)进入宿主细胞。出口途径对寄生虫来说是必不可少的 存活，使该途径的成员具有潜在的药物靶点。ITS的复杂性和广度 寄主细胞重塑机制使恶性疟原虫成为研究寄主病原体的丰富而令人兴奋的系统 互动。然而，这种寄生虫劫持人类红血球能力背后的许多分子机制 血细胞仍然是个谜，因为大部分恶性疟原虫蛋白质组已被证明对结构和 使用传统的重组方法进行生化鉴定。拟议工作的目标是 利用和发展单粒子低温电子显微镜和低温聚焦离子的最新进展 电子束原位冷冻电子断层扫描解释效应器的分子机制 恶性疟原虫蛋白质输出及其抗疟新药结构设计的研究 治疗学。为了实现这些目标，提出了三个目标：1)建立体外转位活性 一种新的必需膜蛋白--出口蛋白疟原虫转位蛋白(PTEX)的检测 复合体，所有输出的效应蛋白都必须通过它才能到达宿主细胞胞浆。这个 已建立的检测方法将使蛋白质易位的分子机制能够进行生化表征 以及通过PTEX抑制剂的结构指导设计获得的抑制剂的筛选。2)结构确定 恶性疟原虫出口组新蛋白复合体的研究。3)超分子效应器的直接可视化 恶性疟原虫感染红细胞中宿主-病原体界面的原位蛋白质输出机制。这个 拟议的工作将提供对这种致命疾病的发病机制的洞察，确定新的疟疾药物靶点， 并使新型抗疟疾疗法的结构导向设计成为可能。

Project Summary Malaria is a devastating parasitic disease that affects more than 200 million people annually, resulting in nearly 500,000 deaths each year. As of 2018, the World Health Organization estimates that 3.8 billion people, roughly half the world's population, are at risk of contracting malaria, and the rise of drug-resistant parasites has created a desperate need for new anti-malarial drugs. While most intracellular pathogens export a limited repertoire of effector proteins to co-opt existing host-cell metabolic machineries, the malaria-causing parasite Plasmodium falciparum exports more than 10% of its proteome into its host, the human red blood cell, during the blood stages of its life cycle. The hundreds of proteins in the P. falciparum exportome extensively remodel host erythrocytes, creating the infrastructure needed to import nutrients, export waste, and evade the host immune system. The export of these hundreds of proteins is complicated by the fact that the malaria parasite conceals itself inside a parasitophorous vacuole (PV) derived from invagination of the host cell plasma membrane during invasion. Following secretion into the PV, proteins destined for export must be unfolded and transported across the PV membrane (PVM) into the host cell in an ATP-dependent process. The export pathway is essential for parasite survival, making members of the pathway attractive potential drug targets. The complexity and breadth of its host-cell remodeling machinery make P. falciparum a rich and exciting system for the study of host-pathogen interactions. However, many of the molecular mechanisms underlying this parasite's ability to hijack human red blood cells remain enigmatic, as much of the P. falciparum proteome has proven recalcitrant to structural and biochemical characterization using traditional recombinant approaches. The goal of the proposed work is to leverage and build upon the latest advances in single-particle cryo electron microscopy and cryo focused ion beam-enabled in situ cryo electron tomography to elucidate the molecular mechanisms underlying effector protein export in P. falciparum and to identify promising targets for structure-based design of new anti-malarial therapeutics. Three aims are proposed to accomplish these goals: 1) Establish an in vitro translocation activity assay for the Plasmodium Translocon of Exported Proteins (PTEX), a novel and essential membrane protein complex, through which all exported effector proteins must pass in order to reach the host cell cytosol. The established assay will enable biochemical characterization of the molecular mechanism of protein translocation and screening of inhibitors obtained via structure-guided design of PTEX inhibitors. 2) Structure determination of novel protein complexes of the P. falciparum exportome. 3) Direct visualization of the supramolecular effector protein export machinery in situ at the host-pathogen interface in P. falciparum-infected erythrocytes. The proposed work will provide insight into the pathogenesis of this deadly disease, identify new malarial drug targets, and enable structure-guided design of novel anti-malarial therapeutics.