Defining the role of TgATG9 in Toxoplasma gondii autophagy and persistence

定义 TgATG9 在弓形虫自噬和持久性中的作用

• 批准号: 10733426
• 负责人: Pariyamon Thaprawat
• 金额: $ 3.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10733426
• 关键词: Acute; Autophagocytosis; Autophagosome; Biogenesis; Biological Assay; Biological Models; Biology; Brain; Cell physiology; Cells; Chronic; Complement; Complex; Cyst; Development; Disease; Eating; Eukaryotic Cell; Event; Eye; Eye diseases; Goals; Heart; Heart Diseases; Homeostasis; Homologous Gene; Homologous Protein; Human; Imaging Techniques; Immune; Immune System Diseases; Immunoprecipitation; Individual; Knock-out; Knowledge; Light; Lipids; Measures; Mediating; Membrane; Microscopy; Molecular; Molecular and Cellular Biology; Mus; Organelles; Organism; Parasites; Pathogenesis; Pathway interactions; Patients; Persons; Physicians; Play; Population; Process; Proteins; Publishing; Recurrence; Recycling; Reporting; Research; Resolution; Respiratory Disease; Risk; Role; Scientist; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Training; Vertebrates; Yeasts; chronic infection; defined contribution; effective therapy; ineffective therapies; insight; interest; knock-down; microscopic imaging; model organism; mutant; nervous system disorder; novel; protein complex; recruit; virtual

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.

项目摘要/摘要 弓形虫是一种广泛存在于细胞内的原生动物寄生虫，它可以感染几乎所有的有核细胞。 包括人类在内的温血脊椎动物。快速生长的急性期寄生虫引起的疾病有限 在其他健康宿主中，然而T.弓形虫有效地转化为缓慢生长的形式，称为缓殖子， 作为细胞内组织囊肿长期存在。据估计，全球人口的三分之一是 慢性感染T.弓形虫，使这些人在大脑，心脏，眼睛， 和其他组织。介导寄生虫持久性的细胞过程在很大程度上是未知的。没有 这些知识阻碍了预防疾病复发措施的战略发展。自慢性期 缓殖子生长非常缓慢，我们认为它们长期依赖于细胞内稳态机制 长期生存。自噬（“自食”）是真核细胞中回收物质和维持生命的重要途径。 细胞内稳态虽然最近已经表明缺乏自噬蛋白TgATG 9的缓殖子， 在慢性感染的小鼠中显示出减少的自噬，具有较低的生存能力，并且产生明显较少的囊肿， 该途径的确切分子机制或动力学仍然是难以捉摸的。该提案的长期目标是 理解和表征T.刚地。 本项目的目标是确定TgATG 9对寄生虫自噬的贡献，沿着鉴定 TgATG 9和整个途径的独特特征。我的建议的具体目标是：1）确定 TgATG 9在T.弓形虫自噬，2）确定的机制和作用， TgATG 9在自噬体生物发生中的作用，以及3）鉴定TgATG 9和膜的相互作用伴侣 T.刚地。在第一个目标下，我将探讨有条件地击倒 TgATG 9对自噬体动力学和自噬货物向寄生虫消化细胞器的递送的影响。到 为此，我将用荧光标记物标记感兴趣的蛋白质，并使用高分辨率点阵光片显微镜 来捕捉自噬途径的动力学。在第二个目标下，我将探讨TgATG 9的作用机制 作为一种潜在的脂质乱序酶由于同源性酵母ATG 9，其功能作为一个scramblase，我将评估 通过酵母互补测定，TgATG 9在敲除菌株中拯救自噬功能的能力。下 第三个目标，我将利用靶向免疫沉淀来鉴定TgATG 9的相互作用伴侣，从而 更好地表征T.弓形虫自噬这项建议完成后，将 为早期分支真核生物的自噬途径提供了新的和基本的见解， 作为T.刚地。它将增加我们对寄生虫体内平衡的途径的理解， 在慢性感染期间被选择性地靶向。它也将作为一个平台，以实现我的培训目标， 实验分子和细胞生物学沿着为我的长期目标提供必要的培训 作为一个物理学家和科学家。