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 most otherwise healthy hosts, however T. gondii efficiently converts to the slow-growing form called bradyzoites that reside long-term within their hosts 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 the 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. Prior studies have shown that T. gondii bradyzoites lacking functional cathepsin L protease activity within the lysosome-like Vacuolar Compartment (VAC) accumulate undigested autophagic material in the VAC. Additionally, bradyzoites deficient in an autophagy protein, TgATG9, show reduced autophagy, have lower viability, and produce markedly fewer cysts in chronically infected mice. In model systems, ATG9 is a core protein in the membrane elongation complex of the autophagy pathway. We seek to define the precise contribution of TgATG9 to autophagy in T. gondii along with identifying unique features of TgATG9 and the pathway as a whole. Our initial results showing that TgATG9 is associated with the VAC and with TgATG8-positive autophagosomes link this protein to the biogenesis and degradation of autophagosomes. Given that T. gondii is an early branching eukaryote, many components of the autophagy pathway are likely to be different from those found in yeast or mammalian cells. To identify novel components of the T. gondii autophagy pathway, we will utilize a proteomics discovery pipeline to identify partners of TgATG9 and other putative ATGs. The identified proteins will be interrogated for their contributions to parasite autophagy along with the establishment and maintenance of tissue cysts in a mouse model of chronic toxoplasmosis.
