Abstract
Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity, but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses of the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, the deletion of CS1 resulted in a significantly reduced flux of glucose-derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than a single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites or there are other routes of citrate supply in the parasite mitochondrion.