Elucidating the role of a mitochondrial superoxide dismutase in Toxoplasma gondii

Toxoplasma gondii is the only apicomplexan parasite that possesses a well-developed reactive oxygen species (ROS) scavenging system, which includes an endogenous catalase and complete glutathione and thioredoxin pathways. Our previous studies generating auranofin-resistant T. gondii parasites led to mutations in T. gondii"s mitochondrial superoxide dismutase (TgSOD2). These parasites showed reduced accumulation of ROS upon exposure to auranofin. TgSOD2 is an essential gene, as evidenced by its low CRISPR-Cas phenotype index and non-viability of constitutive knockouts. During stages of extracellular stress, TgSOD2 is upregulated as the parasite attempts to survive. To understand the essential role of TgSOD2 in T. gondii, we generated knockdown mutants using an auxin-inducible degron system that allowed us to explore the role of TgSOD2 in parasite replication and replication fitness. Its depletion leads to a reduction in replication fitness as these parasites showed decreased plaque formation when compared to their parental line. TgSOD2 depletion led to a reduced number of parasites per parasitophorous vacuole. At the mitochondrial level, depletion of TgSOD2 results in aberrancies in the parasite"s mitochondrion as well as reduction of the parasite"s mitochondrial membrane potential. Through a proximal biotinylation approach, we found that TgSOD2 localizes adjacent to complexes IV and V of the T. gondii mitochondrial electron transport chain, suggesting that these may be sites of ROS generation or ROS-sensitive enzyme subunits. Our studies are the first ones to demonstrate the role of TgSOD2 in parasite life and mitochondrion maintenance.