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， 每年有50万人死亡。截至2018年，世界卫生组织估计有38亿人， 世界上一半的人口都有感染疟疾的危险，抗药性寄生虫的增加已经造成了 迫切需要新的抗疟疾药物。虽然大多数细胞内病原体输出有限的 效应蛋白，以配合现有的宿主细胞代谢机制，疟疾引起的寄生虫疟原虫 在血液阶段，恶性疟原虫将其10%以上的蛋白质组输出到其宿主--人类红细胞中 它的生命周期。恶性疟原虫输出组中的数百种蛋白质广泛重塑宿主红细胞， 创造了输入营养、输出废物和逃避宿主免疫系统所需的基础设施。的 这数百种蛋白质的输出是复杂的，因为疟疾寄生虫隐藏在一个 在入侵过程中，寄主细胞质膜内陷形成的吸寄空泡（PV）。 在分泌到PV中之后，预定用于输出的蛋白质必须解折叠并穿过PV运输 在ATP依赖性过程中，PVM进入宿主细胞。输出途径是寄生虫 生存，使成员的途径有吸引力的潜在药物靶点。其复杂性和广度 恶性疟原虫的宿主细胞重塑机制使其成为研究宿主-病原体关系的一个丰富而激动人心的系统 交互.然而，这种寄生虫能够劫持人类红细胞的许多分子机制， 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是结构和功能上不一致的。 使用传统的重组方法进行生物化学表征。拟议工作的目标是 利用和建立在单粒子低温电子显微镜和低温聚焦离子显微镜的最新进展 束流使能原位低温电子断层扫描，以阐明效应器的分子机制 恶性疟原虫中的蛋白质输出，并确定基于结构设计新的抗疟疾药物的有希望的靶点。 治疗学提出了三个目标来实现这些目标：1）建立体外易位活性 一种新的和必需的膜蛋白--疟原虫输出蛋白质转位子（PTEX）的测定 所有输出的效应蛋白必须通过该复合物才能到达宿主细胞胞质溶胶。的 已建立的测定将能够对蛋白质易位的分子机制进行生物化学表征 以及通过PTEX抑制剂的结构导向设计获得的抑制剂的筛选。2)结构测定 恶性疟原虫输出基因组的新型蛋白质复合物。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。的 拟议的工作将深入了解这种致命疾病的发病机制，确定新的疟疾药物靶点， 并且能够实现新型抗疟疾治疗剂的结构导向设计。

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.