The Role of Toxoplasma Rhoptries in Host Cell Infection

描述(申请人提供)：弓形虫是一种专性细胞内寄生虫，在免疫功能低下的人和先天性感染的新生儿中会导致严重疾病。弓形虫也是研究相关顶复合体寄生虫的模式系统，包括疟疾的病原体恶性疟原虫。这些生物的细胞内生存严重依赖于寄生虫主动入侵宿主细胞、建立允许复制的液泡和避免宿主细胞防御的能力。Apicomplexan寄生虫共有一种独特的入侵机制，包括在入侵的寄生虫和宿主细胞之间形成一个紧密的连接，称为移动连接(MJ)。MJ被认为是寄生虫入侵宿主细胞的稳定锚，也是修改新生液泡的分子筛，使其不与宿主内吞途径融合。弓形虫MJ由寄生虫表面的微粒AMA1组成，与注射到宿主细胞中的棒状体颈蛋白(rons 2/4/5/8)的大分子复合体相连。虽然RON2跨越宿主膜并与AMA1建立联系，但令人惊讶的是，复合体的其余成员位于宿主膜的细胞质表面，它们如何在入侵中发挥作用在很大程度上尚不清楚。我们已经破坏了球虫特异性成分RON8，并表明虽然不是必需的，但该蛋白在体外寄生虫入侵和体内毒力方面发挥着重要作用。由于其余的成员在顶端复合体中保守，并被认为是必需的，这表明顶端复合体包含一个保守的核心复合体，这是入侵所需的，它通过RON8在球虫中得到增强。这一点得到了我们最近对RON5的条件敲除的支持，这表明该蛋白在复合体的组装中起着关键作用，MJ复合体确实是入侵所必需的。我们的第一个目标是 续签申请是对MJ综合体进行深入的功能分析，并确定其架构。具体地说，我们首先将重点放在RON8上，以确定该组件如何增强侵袭能力并将复合体与宿主细胞联系起来。然后，我们将利用RON5的条件敲除来研究它在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比和确定其组成蛋白的关键相互作用来探索该复合体的结构。这些研究将为顶端复合体寄生虫利用这种新的入侵机制感染它们的哺乳动物宿主并导致疾病的机制提供全新的见解。 与公共卫生相关：弓形虫是一种细胞内寄生虫，感染多达三分之一的世界人口，并在免疫功能低下的患者和新生儿中导致严重疾病或死亡。弓形虫也是一些相关寄生虫的模式系统，包括疟疾的病原体恶性疟原虫。这个项目的重点是从寄生虫分泌的一种蛋白质复合体，这些蛋白质是顶复合体寄生虫入侵宿主细胞的关键。由于这种进入宿主细胞的方法是这些寄生虫独有的，是生存所必需的，它代表了治疗干预的新靶点。

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. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is an intracellular parasite that infects up to a third of the world's human population and causes serious disease or death in immunocompromised patients and neonates. T. gondii also serves as a model system for a number of related parasites including Plasmodium falciparum, the causative agent of malaria. This project is focused on a complex of proteins that are secreted from the parasite and are critical for apicomplexan parasites to invade their host cells. As this method of host cell entry i unique to these parasites and essential for survival, it represents a novel target for therapeutic intervention.