Metabolic changes accompanying the loss of fumarate hydratase and malate–quinone oxidoreductase in the asexual blood stage of Plasmodium falciparum

In the glucose-rich milieu of red blood cells, asexually replicating malarial parasites mainly rely on glycolysis for ATP production, with limited carbon flux through the mitochondrial tricarboxylic acid (TCA) cycle. By contrast, gametocytes and mosquito-stage parasites exhibit an increased dependence on the TCA cycle and oxidative phosphorylation for more economical energy generation. Prior genetic studies supported these stage-specific metabolic preferences by revealing that six of eight TCA cycle enzymes are completely dispensable during the asexual blood stages of Plasmodium falciparum, with only fumarate hydratase (FH) and malate-quinone oxidoreductase (MQO) being refractory to deletion. Several hypotheses have been put forth to explain the possible essentiality of FH and MQO, including their participation in a malate shuttle between the mitochondrial matrix and the cytosol. However, using newer genetic techniques like CRISPR and dimerizable Cre, we were able to generate deletion strains of FH and MQO in P. falciparum. We employed metabolomic analyses to characterize a double knockout mutant of FH and MQO (ΔFM) and identified changes in purine salvage and urea cycle metabolism that may help to limit fumarate accumulation. Correspondingly, we found that the ΔFM mutant was more sensitive to exogenous fumarate, which is known to cause toxicity by modifying and inactivating proteins and metabolites. Overall, our data indicate that P. falciparum is able to adequately compensate for the loss of FH and MQO, rendering them unsuitable targets for drug development.