The Role of Toxoplasma Rhoptries in Host Cell Infection

弓形虫在宿主细胞感染中的作用

基本信息

批准号：
8792359
负责人：
Peter John Bradley
金额：
$ 37.79万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-12-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8792359
关键词：
Animals Apicomplexa Architecture Bacterial Adhesins Binding Biological Models Cell membrane Cells Cellular biology Cessation of life Chemicals Coccidia Coccidiosis Complex Coupled Development Disease Eimeria tenella Elements Event Face Falciparum Malaria Funding Grant Human Immunocompromised Host In Vitro Individual Infection Integral Membrane Protein Invaded Knock-out Link Macromolecular Complexes Malaria Maps Mass Spectrum Analysis Mediating Medical Membrane Methods Molecular Motor Names Neck Neospora caninum Organelles Organism Parasites Pathway interactions Plasmodium falciparum Play Population Process Proteins Role Shapes Site Structure Surface Therapeutic Intervention Tight Junctions Toxoplasma Toxoplasma gondii Toxoplasmosis Vacuole Virulence Work Yeasts crosslink design driving force gel electrophoresis human MST1R protein in vivo insight member molecular sieving neonate novel novel therapeutics obligate intracellular parasite parasite invasion pathogen protein complex rhoptry scaffold stoichiometry tool yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in immunocompromised individuals and congenitally infected neonates. Toxoplasma also serves as a model system for the study of related apicomplexan parasites including Plasmodium falciparum, the causative agent of malaria. Intracellular survival of these organisms is critically dependent on the ability of the parasite to actively invade their host cell, establish a replication-permissive vacuole, and avoid host cell defenses. Apicomplexan parasites share a unique mechanism for invasion that involves the formation of a tight junction between the invading parasite and the host cell called the moving junction (MJ). The MJ is believed to form a stable anchor for the parasite to invade the host cell and also serve as a molecular sieve that modifies the nascent vacuole to render it non-fusogenic with the host endocytic pathway. The Toxoplasma MJ consists of micronemal AMA1 on the parasite's surface connected to a macromolecular complex of rhoptry neck proteins (RONs 2/4/5/8) that are injected into the host cell. While RON2 spans the host membrane and establishes the link to AMA1, the remaining members of the complex are surprisingly on the cytoplasmic face of the host membrane and how they function in invasion is largely unknown. We have disrupted the coccidial-specific component RON8 and shown that while not essential, this protein plays an important role in parasite invasion in vitro and in virulence in vivo. As the remaining members are conserved in the Apicomplexa and believed to be essential, this indicates that apicomplexans contain a conserved core complex that is required for invasion, which is enhanced in the coccidia via RON8. This is supported by our recent development of a conditional knockout for RON5, which shows that this protein plays a critical role in assembly of the complex and that the MJ complex is indeed essential for invasion. Our objectives in this first renewal application are to conduct an in depth functional analysis of the MJ complex and determine its architecture. Specifically, we will first focus on RON8 to determine how this component enhances invasion and links the complex to the host cell. We will then exploit the conditional knockout of RON5 to study its role in the organization and function of the MJ complex. Lastly, we will explore the architecture of the complex by determining its stoichiometry and identifying key interactions of its component proteins. These studies will open completely new insight into the mechanism by which apicomplexan parasites use this novel invasion machine to infect their mammalian hosts and cause disease.

描述（由申请人提供）：弓形虫是一种专性细胞内寄生虫，会在免疫功能低下的个体和先天感染的新生儿中引起严重的疾病。弓形虫还可以作为研究相关顶复门寄生虫的模型系统，包括恶性疟原虫（疟疾的病原体）。这些生物体的细胞内生存严重依赖于寄生虫主动侵入宿主细胞、建立允许复制的液泡并避开宿主细胞防御的能力。顶复门寄生虫具有独特的入侵机制，涉及入侵寄生虫与宿主细胞之间形成紧密连接，称为移动连接（MJ）。 MJ被认为为寄生虫侵入宿主细胞形成了稳定的锚点，并且还充当分子筛，修饰新生液泡以使其与宿主内吞途径不融合。弓形虫 MJ 由寄生虫表面的微线 AMA1 组成，该微线 AMA1 连接到注射到宿主细胞中的棒状体颈蛋白 (RONs 2/4/5/8) 大分子复合物。虽然 RON2 跨越宿主膜并与 AMA1 建立联系，但该复合体的其余成员令人惊讶地位于宿主膜的细胞质表面，并且它们在入侵中如何发挥作用很大程度上未知。我们破坏了球虫特异性成分 RON8，并表明该蛋白虽然不是必需的，但在体外寄生虫入侵和体内毒力方面发挥着重要作用。由于其余成员在顶复门中是保守的并且被认为是必需的，这表明顶复门含有入侵所需的保守核心复合物，该复合物在球虫中通过 RON8 得到增强。我们最近开发的 RON5 条件敲除技术支持了这一点，这表明该蛋白在复合物的组装中发挥着关键作用，并且 MJ 复合物确实对于入侵至关重要。我们的目标是第一个更新申请的目的是对 MJ 综合体进行深入的功能分析并确定其架构。具体来说，我们将首先关注 RON8，以确定该成分如何增强侵袭并将复合物与宿主细胞联系起来。然后，我们将利用 RON5 的条件敲除来研究其在 MJ 复合体的组织和功能中的作用。最后，我们将通过确定其化学计量并识别其组成蛋白的关键相互作用来探索复合物的结构。这些研究将为顶复门寄生虫利用这种新型入侵机器感染哺乳动物宿主并引起疾病的机制提供全新的见解。