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Abstract
Toxoplasma gondii is a member of the apicomplexan phylum, a group of parasites that poses a dire threat to global health. While treatments exist for many of these parasites, increasing rates of drug resistance necessitate novel therapeutic development. An organelle that proves a promising target for such development is the T. gondii mitochondrion: it is essential for parasite survival and contains a high number of parasite-specific proteins. A key part of the mitochondrion is the ATP synthase, an enzyme that generates energy in the form of ATP. Intriguingly, over half of T. gondii ATP synthase subunits are apicomplexan-specific. We characterized two divergent ATP synthase subunits containing coiled-coil-helix-coiled-coil-helix (CHCH) domains: ATPTG8 and ATPTG9. In other organisms, CHCH domain proteins play roles in electron transport chain function and mitochondrial morphology. However, ATP synthase-associated CHCH domain proteins have not previously been characterized. We found that ATPTG8 and ATPTG9 are essential for parasite survival and play critical roles in ATP synthase stability and function. Additionally, their loss leads to detrimental effects on multiple aspects of mitochondrial morphology and mutation of key CHCH domain residues disrupted mitochondrial localization. Our work suggests that ATPTG8 and ATPTG9 act as additional scaffolding to stabilize the exceptionally large T. gondii ATP synthase. Though we have gained some insight into its divergent subunits, we know little about how the T. gondii ATP synthase is regulated. Consequently, we characterized the T. gondii homolog of ATPase inhibitory factor 1 (IF1), a conserved ATP synthase inhibitor that can induce cytoprotective gene expression pathways through a process called mitohormesis. While we found that TgIF1 knockout and overexpression did not affect parasite growth under normal conditions and had no impact on metabolism, we did find that knockout reduced cristae density. Furthermore, TgIF1 levels were important for responding to hypoxia, antioxidant expression, gene expression regulation, and recovery from oxidative stress. Although TgIF1 does not appear to participate in metabolic regulation, it seems to be important for stress responses. Ultimately, our work provides insight into the roles of divergent T. gondii ATP synthase subunits and reveals how TgIF1 plays key roles in parasite adaptation to various stressors.