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 i...