Functional Analysis of the T. gondii AMA1 Cytosolic Tail

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Toxoplasma gondii is a widespread protozoan parasite that causes debilitating and sometimes life- threatening disease during pregnancy and in immunocompromised individuals, including those with AIDS. Toxoplasmosis is the second most common cause of death by foodborne illness in the USA and costs the economy billions of dollars annually. T. gondii is also a valuable experimental model for related apicomplexan parasites, including those that cause malaria and cryptosporidiosis. Host cell invasion is essential to the parasite's life cycle, and apical membrane antigen1 (AMA1) is a highly conserved transmembrane protein on the parasite surface that plays an important role in invasion. Much is known about how the extracellular domain of AMA1 interacts with its ligand on the host cell surface (RON2) during invasion, but surprisingly little is known about the function of its short, C-terminal tail, which is located in the parasite cytosol. The central hypothesis of this project is that the cytosolic tail of T. gondii AMA1 (TgAMA1) functions in invasion-related signaling pathways, through interaction with other parasite proteins. The Aims of the project are to: (a) Determine the phenotypic consequences of specific mutations in the TgAMA1 cytosolic tail, establishing at what step(s) in invasion the cytosolic tail functions and the specific residues involved; (b) Identify and determine the biological function of proteins that interact with the TgAMA1 cytosolic tail, establishing whether tail mutations that disrupt invasion also affect the binding of specific proteins; and (c) Determine whether the binding of TgAMA1 to TgRON2 triggers intracellular signaling mediated by the TgAMA1 cytosolic tail, resulting in invasion-related changes to the parasite phosphoproteome. These studies address a significant gap in our understanding of the role that AMA1 plays in the invasion of host cells by apicomplexan parasites. A more complete understanding of AMA1 function will generate new approaches to preventing or controlling the devastating diseases caused by this important group of human pathogens.