Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC₆(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the F_o-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.The apicomplexan parasite Toxoplasma gondii contains a single mitochondrion of an elongated tubular structure (28, 32), which shows several significant metabolic differences from the mammalian counterpart (see references 24 and 33 for review). Although the T. gondii mitochondrion harbors the complete set of enzymes for the tricarboxylic acid cycle (15), it lacks the pyruvate dehydrogenase complex (7, 14, 18), which is typically a central enzyme in carbohydrate metabolism that catalyzes the decarboxylation from pyruvate to acetyl coenzyme A. Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33). The T. gondii genome predicts the presence of all components necessary for a respiratory chain. Biochemical evidence for oxidative phosphorylation was provided by extracellular T. gondii tachyzoites that were permeabilized with digitonin (39). However, the overall contribution of oxidative phosphorylation to energy production in relation to other ATP-generating pathways has not been satisfactorily clarified for intracellular T. gondii so far.A fundamental difference of the T. gondii and also the Plasmodium falciparum electron transport chains (ETCs) as opposed to the mammalian ETC is the lack of multisubunit complex I, which couples the transfer of electrons from NADH to ubiquinone with the translocation of protons (6). Instead, P. falciparum expresses one isoform (2) and T. gondii expresses two isoforms (22) of so-called “alternative” or type II NADH dehydrogenases (NDH2s). These single-subunit enzymes do not transport protons across the membrane, and they are, in contrast to the NADH-oxidizing activity of complex I, not rotenone sensitive (21, 27). NDH2s can occur in two topological orientations with respect to the inner mitochondrial membrane. Internal enzymes are facing with their active site toward the mitochondrial matrix and use mitochondrial NAD(P)H as the electron donor, while external enzymes use cytosolic NAD(P)H. Up to now, the orientation of the apicomplexan isoforms is unknown.Due to their absence in the mammalian host, NDH2s were proposed to be promising drug targets against Mycobacterium tuberculosis (40). Their suitability as a drug target in Plasmodium is controversial and has been the subject of discussion (16, 17, 38). Previously, it was demonstrated that low-affinity NDH2 inhibitors in micromolar concentrations were able to inhibit the activity of the P. falciparum NDH2 and led to a collapse of the mitochondrial membrane potential (ΔΨm) (2). The only high-affinity NDH2 inhibitors described so far are 1-hydroxy-2-alkyl-4(1H)quinolones with long alkyl-site chains, for example, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) (C₁₂), which possesses structural similarity to ubiquinone. These compounds were shown to inhibit the activities of Yarrowia lipolytica NDH2 (13) and T. gondii NDH2-I (22) with 50% inhibitory concentrations of between 200 and 300 nM. HDQ was also shown to effectively inhibit T. gondii and P. falciparum replication in nanomolar concentrations in tissue cultures (31).We demonstrate in this study that HDQ treatment in nanomolar concentrations leads to a depolarization of the T. gondii ΔΨm within minutes. The subsequent lack of oxidative phosphorylation leads to a ∼70% reduction of the intracellular ATP level within 24 h. This suggests an indispensable role of NDH2 activity in the maintenance of the ΔΨm and in energy metabolism in the tachyzoite stage.