Variation among Plasmodium falciparum Strains in Their Reliance on Mitochondrial Electron Transport Chain Function

Abstract

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short-or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.With greater than 3 billion people at risk, ϳ240 million people infected, and nearly 1 million deaths in 2008, malaria remains one of the world's leading killers (38). Plasmodium falciparum is the most lethal among the species causing human infections. Asexual blood-stage parasites of P. falciparum contain a single mitochondrion with minimal, but essential, physiological functions (22,34). The mitochondrial electron transport chain (mtETC) is the primary generator of the proton electrochemical gradient (⌬p) across the mitochondrial inner membrane. While ⌬p does not appear to power mitochondrial ATP synthesis in blood stages of P. falciparum, its establishment is vital to the parasite, and it is required to transport metabolites and proteins in and out of the mitochondrion (20,25,30). Another critical function of the malaria mtETC is the reoxidation of dihydroubiquinone (CoQH 2 ) to ubiquinone (CoQ), which is the electron acceptor for several mitochondrial dehydrogenases, such as dihydroorotate dehydrogenase (DHODH), glycerol 3-phosphate dehydrogenase (GPDH), succinate dehydrogenase (SDH), type 2 NADH dehydrogenase (NDH2), and malate-quinone oxidoreductase (MQO) (10,12,20,21,25). DHODH catalyzes the fourth step and the s...

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“…Thus, yDHODH transgenic lines have become a convenient tool for determining if a compound targets the mtETC. Here, we utilized the 3D7attB-yDHODH line, which has the yDHODH gene integrated into the genome at a nonessential locus (43). As shown in Fig.…”

“…The mtETC is an essential process in malaria parasites and a known antimalarial drug target. We have shown that in the asexual blood stages, the critical function of the mtETC is to sustain the activity of the parasite DHODH for pyrimidine biosynthesis (35,43). Provision of the parasites with the yDHODH, which does not rely on the mtETC, makes the parasites resistant to all mtETC inhibitors (35,43).…”

“…P. falciparum strains Dd2 (resistant to chloroquine, mefloquine, and pyrimethamine) and 3D7 (drug sensitive) are the wild-type lines used in this study. P. falciparum 3D7attB-yDHODH is a transgenic line bearing a copy of the yDHODH gene in its genome, rendering the parasite resistant to inhibitors targeting the mtETC (35,43). Parasites were cultured in human O ϩ erythrocytes (Interstate Blood Bank) in complete RPMI 1640 medium supplemented with 0.5% AlbuMAX (Invitrogen), 15 mM HEPES, 10 mg/liter hypoxanthine, 25 mM NaHCO 3 , and 50 g/liter gentamicin.…”

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…do require mitochondrial metabolism because they are susceptible to drugs targeting the ETC, such as atovaquone[ 1 2 _ T D $ D I F F ] , that inhibits complex III [32][33][34][35][36][37]. Evidently this is not for ATP synthesis because inhibition of the ETC has little effect on cellular ATP levels [38][39][40], and deletion of the functional b-subunit of ATP synthase can be tolerated in blood stages of P. berghei [31].…”

Atovaquone Tolerance in Plasmodium falciparum Parasites Selected for High-Level Resistance to a Dihydroorotate Dehydrogenase Inhibitor

“…1B and Table 1). This phenotype is not like the full resistance seen in traditional studies (12)(13)(14) because proliferation stalls at ϳ50%, and parasites persist at a low level even at atovaquone concentrations as high as 10 M in a 72-h study (as measured by SYBR Green DNA labeling or by hypoxanthine uptake assays) ( Table 1 and Fig. 1B).…”