The Role of Toxoplasma Rhoptries in Host Cell Infection

描述（由申请方提供）：弓形虫是一种专性细胞内寄生虫，可导致免疫功能低下个体和先天性感染新生儿发生严重疾病。弓形虫也是研究相关顶复门寄生虫（包括疟疾病原体恶性疟原虫）的模型系统。这些生物体的细胞内存活严重依赖于寄生虫主动侵入其宿主细胞、建立允许复制的空泡和避免宿主细胞防御的能力。 顶复门寄生虫具有独特的入侵机制，包括在入侵寄生虫和宿主细胞之间形成称为移动连接（MJ）的紧密连接。MJ被认为形成寄生虫入侵宿主细胞的稳定锚，并且还充当修饰新生液泡以使其与宿主内吞途径非融合的分子筛。弓形虫MJ由寄生虫表面上的微线AMA 1组成，其连接到注射到宿主细胞中的棒状体颈蛋白（RON 2/4/5/8）的大分子复合物。虽然RON 2跨越宿主膜并建立与AMA 1的连接，但复合物的其余成员令人惊讶地位于宿主膜的细胞质面上，并且它们在入侵中如何发挥作用在很大程度上是未知的。我们已经破坏了球虫特异性组分RON 8，并表明虽然不是必需的，但这种蛋白质在体外寄生虫入侵和体内毒力中起着重要作用。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，其在球虫中通过RON 8增强。我们最近开发的RON 5的条件性敲除支持了这一点，这表明这种蛋白质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。 我们的目标首先是 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RON 8，以确定该组分如何增强入侵并将复合物与宿主细胞联系起来。然后，我们将利用RON 5的条件性敲除来研究其在MJ复合物的组织和功能中的作用。最后，我们将通过确定其化学计量和确定其组分蛋白质的关键相互作用来探索复合物的结构。这些研究将为了解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。

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.