Defining the role of TgATG9 in Toxoplasma gondii autophagy and persistence

项目摘要/摘要 弓形虫是一种普遍存在于细胞内的原生动物寄生虫，可感染几乎所有 包括人类在内的温血脊椎动物。寄生虫快速生长的急性期只会引起有限的疾病 然而，在其他健康的宿主中，弓形虫有效地转化为生长缓慢的形式，称为缓殖子 长期以细胞内组织囊肿的形式存在。据估计，全球人口的三分之一是 慢性感染弓形虫，使这些人面临大脑、心脏、眼睛重新激活疾病的风险， 和其他纸巾。调节寄生虫持久性的细胞过程在很大程度上是未知的。不存在 这种认识阻碍了预防复发性疾病的措施的战略性发展。慢性期以来 缓虫体生长非常缓慢，我们认为它们长期依赖细胞内环境平衡机制。 长期生存。自噬(“自噬”)是真核细胞回收物质和维持生命的重要途径。 细胞动态平衡。虽然最近有研究表明，缓虫缺乏一种自噬蛋白TgATG9， 在慢性感染的小鼠中，自噬减少，存活率降低，产生的包囊明显减少， 这一途径的确切分子机制或动力学仍然难以捉摸。这项提议的长期目标是 了解和表征弓形虫自噬途径中涉及的蛋白质的分子机制。 该项目的目标是确定TgATG9对寄生虫自噬的贡献以及识别 TgATG9的独特功能和整个通路。我的建议的具体目的是：1)确定 TgATG9在弓形虫自噬中的定位和募集动态，2)确定TgATG9的机制和作用 TgATG9在自噬小体发生中的作用；3)确定TgATG9与膜的相互作用伙伴 弓形虫的伸长复合体。在第一个目标下，我将探讨有条件淘汰的后果 TgATG9关于自噬体动力学和将自噬货物运送到寄生虫消化器官的研究。至 做到这一点，我将用荧光标记标记感兴趣的蛋白质，并使用高分辨率晶格光片显微镜 来捕捉自噬途径的动态。在第二个目标下，我将探索TgATG9的机制 作为一种潜在的脂类扰乱酶。由于与酵母ATG9同源，它的功能是扰乱酶，我将评估 TgATG9通过酵母互补试验挽救基因敲除菌株自噬功能的能力。在……下面 第三个目标，我将利用定向免疫沉淀来确定TgATG9的相互作用伙伴，从而 更好地描述了弓形虫自噬过程中的膜伸长复合体。这项提案完成后，将 为早期分枝真核生物的自噬途径提供了新的和基本的见解 作为弓形虫。这将增加我们对涉及寄生虫动态平衡的途径的理解，该途径可能 在慢性感染期间有选择地成为目标。它也将成为实现我的培训目标的平台 实验分子和细胞生物学，以及为我的长期目标提供必要的培训 作为一名内科科学家。

PROJECT SUMMARY / ABSTRACT Toxoplasma gondii is a pervasive intracellular protozoan parasite that can infect any nucleated cell in virtually all warm-blooded vertebrates including humans. The fast-growing, acute stage of the parasite causes limited illness in otherwise healthy hosts, however T. gondii efficiently converts to the slow-growing form called bradyzoites that reside long-term as intracellular tissue cysts. It is estimated that one third of the global human population is chronically infected with T. gondii, rendering such individuals at risk for reactivated disease in the brain, heart, eyes, and other tissues. The cellular processes mediating parasite persistence are largely unknown. The absence of such knowledge impedes strategic development of measures to preclude reactivated disease. Since chronic stage bradyzoites grow very slowly, we propose that they shift to relying on cellular homeostatic mechanisms for long term survival. Autophagy (“self-eating”) is an important pathway in eukaryotic cells to recycle materials and maintain cellular homeostasis. While it has recently been shown that bradyzoites deficient in an autophagy protein, TgATG9, show reduced autophagy, have lower viability, and produce markedly fewer cysts in chronically infected mice, the exact molecular mechanisms or dynamics of this pathway remain elusive. The long-term goal of this proposal is to understand and characterize the molecular mechanisms of proteins involved in the autophagy pathway in T. gondii. The objectives of this project are to define the contribution of TgATG9 to parasite autophagy along with identifying unique features of TgATG9 and the pathway as a whole. The specific aims of my proposal are: 1) to determine the localization and recruitment dynamics of TgATG9 in T. gondii autophagy, 2) to define the mechanism and role of TgATG9 in autophagosome biogenesis, and 3) to identify interacting partners of TgATG9 and the membrane elongation complex in T. gondii. Under the first aim, I will explore the consequences of conditional knockdown of TgATG9 on autophagosome dynamics and delivery of autophagic cargo to the parasite’s digestive organelle. To do this, I will tag proteins of interest with a fluorescent marker and use high resolution lattice light-sheet microscopy to capture the dynamics of the autophagy pathway. Under the second aim, I will explore the mechanism of TgATG9 as a potential lipid scramblase. Due to homology to yeast ATG9, which functions as a scramblase, I will evaluate the ability of TgATG9 to rescue autophagy function in a knockout strain via yeast complementation assays. Under the third aim, I will utilize targeted immunoprecipitation to identify the interacting partners of TgATG9 and thereby better characterize the membrane elongation complex in T. gondii autophagy. This proposal, when completed, will provide novel and fundamental insights into the autophagy pathway of early branching eukaryotic organisms such as T. gondii. It will increase our understanding of a pathway involved in parasite homeostasis that could potentially be selectively targeted during chronic infection. It will also serve as a platform to achieve my training goals in experimental molecular and cellular biology along with providing me with requisite training for my long-term goals as a physician-scientist.