The Role of Toxoplasma Rhoptries in Host Cell Infection

描述（由申请人提供）：刚地弓形虫是一种专性细胞内寄生虫，可在免疫功能低下的个体和先天性感染的新生儿中引起严重疾病。弓形虫还可作为研究包括疟疾病原体恶性疟原虫（Plasmodium falciparum）在内的相关顶复合寄生虫的模型系统。这些生物的细胞内存活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和避开宿主细胞防御的能力。顶复合体寄生虫有一种独特的入侵机制，即在入侵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点，也可以作为分子筛来修饰新生液泡，使其与宿主内吞途径不发生融合。弓形虫MJ由寄生体表面的微型AMA1与注入宿主细胞的弓形虫颈部蛋白（ron2/4/5 /8）的大分子复合物连接而成。当RON2跨越宿主膜并与AMA1建立联系时，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，并且它们在入侵中的作用在很大程度上是未知的。我们已经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中起重要作用。由于其余成员在顶复合体中是保守的，并且被认为是必需的，这表明顶复合体含有一个保守的核心复合体，这是入侵所必需的，在球虫中通过RON8增强。我们最近对RON5的条件敲除研究支持了这一点，这表明该蛋白在复合物的组装中起着关键作用，并且MJ复合物确实是入侵所必需的。我们的目标在此第一

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.