UC Irvine

Toxoplasma gondii is the only apicomplexan parasite known to possess a well-developed reactive oxygen species (ROS) scavenging system which includes an endogenous catalase and complete glutathione and thioredoxin pathways. Compared against other apicomplexan parasites, T. gondii is uniquely equipped with a wide array of ROS scavenging systems. These ROS scavenging enzymes are key to neutralizing both endogenous and host cell generated ROS. By virtue of having more ROS scavenger systems, T. gondii may be capable of tolerating a wider variety of intracellular conditions, which is then reflected in its wide host cell tropism. Conversely, these ROS scavenging systems may also be the key to eliminating parasitic infections as their components are unique to the parasite. Several treatment options are able to selectively inhibit the activity of parasite ROS scavengers, and thereby treat various apicomplexan parasitic infections. Of ROS scavenger targeting therapeutic strategies, auranofin was thought to specifically inhibit pathogen thioredoxin reductase. To determine if thioredoxin reductase is still the target of auranofin in T. gondii, we isolated drug resistant mutants. Auranofin resistant T. gondii parasites, harbor mutations in T. gondii’s mitochondrial superoxide dismutase (TgSOD2). These parasites showed reduced accumulation of ROS upon exposure to auranofin. However, the L201P mutation of TgSOD2 is not sufficient to confer resistance to auranofin. Our work indicated that auranofin likely induces parasite death by apoptosis: evidenced by parasite shrinkage and exposure of phosphatidylserine (PS) residues on the parasite surface detected by Annexin V. To understand the role of TgSOD2 throughout the parasite’s life, we generated inducible knockdown mutants using an auxin-inducible degron system that allowed us to explore the role of TgSOD2 in parasite replication and replication fitness (Chapter Three). During stages of extracellular stress, TgSOD2 transcription is upregulated. TgSOD2 depletion leads to reduction in replication fitness as these parasites showed decreased plaque formation when compared to their parental line. At the mitochondrial level, depletion of TgSOD2 results in aberrant mitochondrial morphology 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 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. Lastly, Chapter Four describes the future lines of inquiry opened by the findings presented in Chapters Two and Three. Based on the studies presented, TgSOD2 is likely implicated in the formation or maintenance of the ATP synthase complex. Furthermore, inhibition of TgSOD2 may sensitize the parasite to further ROS damage. Future studies ought to investigate the link between TgSOD2 and ATP synthase, then evaluate the viability of dual inhibition as a therapeutic strategy for T. gondii infection clearance.