Abstract
The mitochondrion of the deadliest human malaria parasite, Plasmodium falciparum, is an essential source of cellular acetyl-CoA during the asexual blood-stage of the parasite life cycle. Blocking mitochondrial acetyl-CoA synthesis leads to a hypoacetylated proteome and parasite death. We previously determined that mitochondrial acetyl-CoA is primarily synthesized from glucose-derived pyruvate by α-ketoacid dehydrogenases. Here, we asked if inhibiting the import of glycolytic pyruvate across the mitochondrial inner membrane would affect acetyl-CoA production and, thus, could be a potential target for antimalarial drug development. We selected the two predicted mitochondrial pyruvate carrier proteins (PfMPC1 and PfMPC2) for genetic knockout and isotopic metabolite tracing via HPLC-MS metabolomic analysis. Surprisingly, we observed that asexual blood-stage parasites could survive the loss of either or both PfMPCs with only minor growth defects, despite a substantial reduction in the amount of glucose-derived isotopic labelling into acetyl-CoA. Furthermore, genetic deletion of two additional mitochondrial carboxylic acid transporters - DTC (di/tricarboxylic acid carrier) and YHM2 (a putative citrate/α-ketoglutarate carrier protein) - only mildly affected asexual blood-stage replication, even in the context of PfMPC deficiency. Although we observed no added impact on the incorporation of glucose carbon into acetyl-CoA in these quadruple knockout mutants, we noted a large decrease in glutamine-derived label in tricarboxylic acid cycle metabolites, suggesting that DTC and YHM2 both import glutamine derivatives into the mitochondrion. Altogether, our results expose redundant routes used to fuel the blood-stage malaria parasite mitochondrion with imported carbon from two major sources - glucose and glutamine.