Autophagy is a highly regulated, critical process that cells rely upon for stress response,differentiation, tissue remodeling, and apoptosis. Autophagy is remarkably well-conserved ineukaryotes ranging from yeast to humans, and some of the core autophagy (ATG) proteins can befound in early-branching eukaryotes such as the pathogenic parasite Toxoplasma gondii.Interestingly, Toxoplasma exhibits some features of canonical autophagy upon amino acid starvation,such as redistribution and lipidation of autophagy protein ATG8 (TgATG8); however, many corecomponents of the autophagy machinery are missing or highly divergent, suggesting that autophagyis streamlined in this parasite or its ATG proteins have unique functions. The observation thatTgATG8 associates with the essential plastid-like organelle known as the apicoplast in a lipidation-dependent manner supports the idea that ATG proteins possess novel functions in Toxoplasma.TgATG8 and other TgATGs are essential for parasite survival, which further warrants their importancefor study, but prohibits the generation of knockout parasites as a tool to understand their biologicalrole. Thus, alternative approaches will be required to interrogate this important class of proteins inToxoplasma. We have recently found that lysine 23 (K23) of TgATG8 is acetylated and hypothesizethat the study of post-translational modifications (PTMs) will yield vital new insights into TgATGproteins. As proof of principle, we mutated K23 to a nonacetylatable amino acid and surprisingly foundspontaneous encystation occurring in mutant parasites. Thus, we have discovered a novel role forTgATG8 in the conversion of replicating parasites to their latent stage. We propose to elucidate themechanism underlying this striking phenotype, thereby filling a knowledge gap linking autophagyhomologues and cyst conversion. To begin deciphering the functionality of TgATG8, we will determinethe timing of its acetylation and monitor autophagic flux, TgATG8 localization, and TgATG8 proteininteractions in K23 mutant parasites. Furthermore, we will determine the role of TgATG8 acetylation inparasite development and virulence through in vivo experiments. To establish a new understanding ofthe role of TgATG8, we will also determine the localization and PTM occurring on the core TgATG8-conjugation complex homologues TgATG3, TgATG4, TgATG5, and TgATG7 in normal versusautophagic conditions. These studies will provide a wealth of new data for the research community,exposing new avenues to investigate canonical and noncanonical roles of the TgATG8-conjugationsystem, which promises to lead to new targets for treatment of toxoplasmosis and related parasiticdiseases.
|Effective start/end date||6/15/16 → 5/31/18|
- National Institutes of Health: $232,600.00