Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages

Abstract

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum , the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P . falciparum , PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P . falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc 1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc 1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.

Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions

“…The Cas9 CDS is flanked by an yDHOD (yeast dihydroorotate dehydrogenase) gene on the 5' end and a Flag tag on the 3' end. We previously removed the yDHOD gene from pAIO, yielding a pre-gRNA construct without yDHOD designated pAIO-Cas9-yDHOD(-) (48). In pAIO-Cas9-yDHOD(-), we performed a series of cloning steps to replace BtgZI with EcoRI for cloning gRNAs and modified the trans-activating sequence of the gRNA according to published studies (59,60) to enhance its binding to Cas9 (details in the Supporting Information).…”

“…Briefly, the progress of a 300 µL of cyt c reductase reaction, which consisted of mitoprep samples (containing bc 1 complex), reduced decylubiquinol (electron donor), oxidized horse heart cytochrome c (electron acceptor) and KCN (inhibiting Complex IV activity) in buffer at pH 7.4, was recorded at 550 nm using the spectrophotometer. Other procedures followed our published studies (36,48).…”

Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution

“…In another study, selection of P. falciparum with putative inhibitors of P. falciparum NADH dehydrogenase 2 (PfNDH2) led to mutations in the more likely target: the parasite cytochrome B gene expressed by the mitochondrial genome (237). Consistent with these findings, deletion of the pfndh2 gene has been shown to be dispensable in asexual blood stages, and further, its deletion does not alter the parasite's susceptibility to multiple mitochondrial electron transport chain (mtETC) inhibitors (241).…”

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

“…Complex II and MQO are components of the TCA cycle, and NDH-2 is required to re-oxidize NADH produced in mitochondrial metabolism, including the TCA cycle. Genetic validation conducted using P. berghei-infected mice or cultured P. falciparum has shown that NDH-2 [9,10] and complex II [11,12] are essential for oocyst development inside the mosquito, while MQO is required (at minimum) in the asexual blood stage [7,13]. Little is known about the role of G3PDH in apicomplexan parasites, although some studies have suggested a role for G3PDH in redox-balance, gluconeogenesis, and lipid biosynthesis [14,15].…”