ATP Synthase‐Associated Proteins TgASAP5 and TgASAP6 Sustain Mitochondrial Homeostasis in Toxoplasma gondii

ABSTRACT Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects a wide range of warm‐blooded animals and causes a globally prevalent zoonotic disease. Mitochondrial ATP synthase is the terminal complex of oxidative phosphorylation and plays an essential role in parasite energy metabolism. However, the functions of many parasite‐specific ATP synthase‐associated proteins remain largely unknown. Here, we identified and characterized two novel ATP synthase‐associated proteins, TgASAP5 and TgASAP6, in T. gondii and generated conditional knockdown parasite strains to investigate their functions. Subcellular localization and protein interaction analyses demonstrated that TgASAP5 and TgASAP6 are mitochondrial proteins associated with the ATP synthase core subunit F 1 β. Functional analyses revealed that depletion of either TgASAP5 or TgASAP6 significantly impaired parasite invasion, intracellular replication, lytic growth, and virulence in mice. Knockdown of TgASAP5 or TgASAP6 disrupted mitochondrial inner membrane organization and was associated with reduced ATP production, elevated reactive oxygen species levels, and reduced mitochondrial membrane potential. Transcriptomic analysis further revealed extensive alterations in mitochondrion‐associated gene expression, affecting oxidative phosphorylation and energy metabolism. Collectively, these findings demonstrate that TgASAP5 and TgASAP6 are essential components of the T. gondii ATP synthase complex and are required for maintaining mitochondrial integrity, energy homeostasis, and parasite survival. This study provides new insights into the molecular regulation of mitochondrial ATP synthase in apicomplexan parasites.