Exploring redundancies in central carbon metabolism in Toxoplasma parasites

Toxoplasma gondii is an apicomplexan parasite that causes severe disease in immunocompromised individuals, newborns, and livestock. This ubiquitous parasite uses central carbon metabolism pathways to generate energy and macromolecules for its proliferation and survival. In other eukaryotes, the mitochondrial tricarboxylic acid (TCA) cycle plays a key role in energy generation and the provision of biosynthetic intermediates. T. gondii harbours a complete functional mitochondrial TCA cycle, but the enzymes that catalyse the individual metabolic reactions remain understudied, and the overall importance of the TCA cycle is still uncertain. To address this knowledge gap, we investigated the importance and role of the TCA cycle in T. gondii, using a combination of forward and reverse genetics as well as physiological and metabolomic analyses. We found that the loss of some TCA cycle reactions led to severe defects in parasite proliferation and mitochondrial oxygen consumption in vitro, whereas loss of others had minimal impact. Using CRISPR/Cas9 genome-wide screening of TCA cycle mutants we discovered the presence of several functional redundancies. One notable redundancy was in the reactions catalysed by the mitochondrial TCA cycle enzyme malate:quinone oxidoreductase (MQO) and the cytosolic enzyme malate dehydrogenase (MDH), both of which mediate malate oxidation but in different subcellular compartments. Parasites which simultaneously lack both MQO and MDH exhibited a defective TCA cycle, impaired pyrimidine biosynthesis and accumulation of fumarate. We are currently investigating the essential metabolic process(es) that require malate oxidation in the parasite. Overall, our findings enhance the understanding of a key metabolic pathway and the flexible nature of central carbon metabolism in T. gondii.