Novel rhoptry effector proteins in Toxoplasma host-pathogen interaction

描述（由申请人提供）：刚地弓形虫是一种专性细胞内寄生虫，在免疫功能低下的个体中引起严重的中枢神经系统疾病，在先天感染的新生儿中引起出生缺陷。这些生物的细胞内存活依赖于入侵宿主细胞、建立允许复制的生态位和避免宿主细胞防御的能力。分泌型细胞已成为调控宿主细胞入侵和劫持功能的关键细胞器。为了劫持宿主功能，异构体向宿主细胞质中注入大量蛋白质，这些蛋白质可以被运输到受感染细胞内的不同区室，并直接靶向宿主途径。这些宿主“效应”蛋白的重要性可以通过注射的一组线状体激酶来证明，这些线状体激酶在调节宿主信号通路和调节寄生虫毒力方面发挥着关键作用。除了ROP激酶，异构体还注入一系列其他功能未知的效应蛋白。这些蛋白大多与已知蛋白或可识别结构域缺乏同源性，是弓形虫和密切相关的寄生虫所特有的。我们已经确定了一组这些新的网状效应物，并在生物学上相关的II型菌株寄生虫中开发了基因敲除。在这个提议中，我们将确定这些新的效应蛋白在调节宿主功能中的作用。具体来说，我们将确定宿主的反应是什么？效应菌株在体外使用宿主微阵列分析，在体内通过评估小鼠的毒力、组织趋向性和慢殖子形成。我们还将使用串联亲和标记效应蛋白在宿主细胞中的外源表达来确定它们的目的地和识别宿主靶标。总之，这些互补的方法有望揭示弓形虫如何利用这套独特的效应蛋白来调节其哺乳动物宿主细胞的新的机制见解。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite that causes severe central nervous system disorders in immunocompromised individuals and birth defects in congenitally infected neonates. Intracellular survival of these organisms is dependent on the ability to invade their host cell, establish a replication-permissive niche, and avoid host cell defenses. The secretory rhoptries have emerged as a key organelle that regulates both invasion and hijacking of host cell functions. For hijacking host functions, the rhoptries inject a burst of proteins into the host cell cytoplasm that can be trafficked to distinct compartments within the infected cell and directly target host pathways. The importance of these host "effector" proteins is exemplified by a family of rhoptry kinases that are injected and play critical roles in modulating host signaling pathways and regulating parasite virulence. In addition to ROP kinases, the rhoptries inject an array of other effector proteins that are of unknown function. Most of these proteins lack homology to known proteins or identifiable domains and are unique to Toxoplasma and closely related parasites. We have identified a panel of these novel rhoptry effectors and developed gene knockouts in the biologically relevant type II strain parasites for their study. In this proposal, we will determine the role of these novel effector proteins in regulating host functions. Specifically, we will determine the host response to ?effector strains in vitro using host microarray analysis and in vivo by assessing virulence, tissue tropism, and bradyzoite formation in mice. We will additionally use exogenous expression of tandem affinity tagged effector proteins in host cells to determine their destination and identify host targets. Together, these complementary approaches promise to reveal novel mechanistic insights into how Toxoplasma uses this unique set of effector proteins to modulate its mammalian host cell.