SUMMARY New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.

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Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc1 complex. As with other cytochrome bc1 inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg–1. In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite’s mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock. spp., the causative agent of malaria, contains a mitochondrion that is very divergent from that of their hosts. The malarial mitochondrion is a clinically validated target for the antimalarial drug atovaquone, which specifically blocks the electron transfer activity of the complex of the mitochondrial electron transport chain (mtETC). Most mtETC proteins are nuclear-encoded and imported from the cytosol, but three key protein subunits are encoded in the mitochondrial genome: cyt, COXI, and COXIII. They are translated inside the mitochondrion by mitochondrial ribosomes (mitoribosomes). Here, we characterize the function of one large mitoribosomal protein in , PfmRPL13. We found that PfmRPL13 localizes to the parasite mitochondrion and is refractory to genetic knockout. Ablation of PfmRPL13 using a conditional knockdown system (TetR-DOZI-aptamer) caused a series of adverse events in the parasite, including mtETC deficiency, loss of mitochondrial membrane potential (Δψ), and death. The PfmRPL13 knockdown parasite also became hypersensitive to proguanil, a drug proposed to target an alternative process for maintaining Δψ Surprisingly, transmission EM revealed that PfmRPL13 disruption also resulted in an unusually elongated and branched mitochondrion. The growth arrest of the knockdown parasite could be rescued with a second copy of PfmRPL13, but not by supplementation with decylubiquinone or addition of a yeast dihydroorotate dehydrogenase gene. In summary, we provide first and direct evidence that mitoribosomes are essential for malaria parasites to maintain the structural and functional integrity of the mitochondrion.