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

Abstract

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 DiOC(6)(3) (3,3'-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (Delta Psi m) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a Delta Psi m collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. Delta Psi m 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 Delta Psi m, since Delta Psi m-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.

FIG. 1.

(A) HDQ treatment decreases the ΔΨm of intracellular parasites. HFFs were infected with RH strain tachyzoites and treated at the indicated time points with 100 nM HDQ for a period of 6 h, followed by Mitotracker staining and fixation. Drug-untreated controls were stained in parallel at the same time points. The fraction of vacuoles containing ΔΨm-positive parasites was determined by fluorescence microscopy of at least 100 vacuoles. Results are expressed as means ± standard deviations (SD) of data from duplicate slides from a representative experiment (n = 2). (B) Comparison of the Mitotracker staining patterns from a sample in which the 6-h HDQ treatment period was started 16 h postinfection (top) to those from an untreated control (bottom). Scale bars, 5 μm.

FIG. 2.

Real-time imaging of the T. gondii ΔΨm by DiOC₆(3) staining. (A) Parasites expressing the mitochondrial marker S9-RFP were stained with the cationic fluorophore DiOC₆(3) at different time points postinfection and analyzed by fluorescence live-cell imaging. DiOC₆(3) specifically stained the mitochondria of the parasites and also the host cell mitochondria, which appear to be less intensely stained than the T. gondii mitochondria. Scale bars, 5 μm. (B) Kinetics showing the influence of 10 nM complex III inhibitor atovaquone (ATO) and 10 nM, 100 nM, and 1 μM NDH2 inhibitor HDQ on the ΔΨm of individual parasites. The infected cultures were stained immediately before drug treatment with DiOC₆(3). Scale bars, 5 μm. (C) Quantification of mitochondrial membrane depolarization kinetics after treatment with 10 nM, 100 nM, and 1 μM HDQ; 10 nM atovaquone (ATV); and a combination of 10 nM HDQ and 10 nM atovaquone. The infected cultures were stained immediately before drug treatment with DiOC₆(3), and the fraction of vacuoles containing ΔΨm-positive parasites was determined by fluorescence microscopy of at least 100 vacuoles at the indicated time periods. The diagram shows the means ± SD of data from duplicate wells from a representative experiment.

FIG. 3.

Substrate supplementation in permeabilized parasites partly decreases HDQ-mediated ΔΨm depolarization. DiOC₆(3)-stained intracellular parasites were digitonin permeabilized and treated with 1 μM HDQ (A) or 1 μM atovaquone (ATO) (B) either alone or in combination with 10 mM malate (MAL); 10 mM succinate (SUC); 10 mM dihydroorotate (DHO); 1 mM glycerol-3-phosphate (G-3-P); 10 mM oxaloacetate (OAA); a mixture of malate, succinate, dihydroorotate, and glycerol-3-phosphate (SUB); and 0.2 mM TMPD-1.5 mM ascorbate (TMPD/ASC). The percentage of ΔΨm-positive parasites was determined from pictures taken by fluorescence microscopy after a 15- to 25-min incubation period at 37°C. Results are expressed as means ± SD of data from duplicate samples from a representative experiment (n = 2). ^, P < 0.002; *, P < 0.005; **, P < 0.03; ***, P < 0.02; #, P < 0.01; ##, P < 0.001 (determined by a Student's t test) (A). *, P < 0.003 (determined by a Student's t test) (B).

FIG. 4.

Inhibition of membrane-associated T. gondii F_oF₁-ATPase attenuates HDQ-mediated ΔΨm depolarization. (A) RH strain tachyzoites stably transfected with pTet7Sag4-TgATP-β-cmyc-DHFR were analyzed by immunofluorescence assay using anti-myc MAb. Mitochondrial localization of myc-tagged ATPase-β was confirmed by colocalization with Mitotracker fluorescence. (B) Parasites expressing myc-tagged ATPase-β were fractionated into a soluble fraction (S) and a membranous fraction (P). Both fractions were separated by SDS-PAGE and analyzed by immunoblotting with an anti-myc antibody. (C) The mitochondrial DNA-lacking cell line 143B/260 was infected with T. gondii RH strain cells and treated at 18 h postinfection with 100 nM HDQ or 1 μM oligomycin for 6 h. Parasites were released from host cells by syringe passage and immediately stained with Mitotracker. The percentage of ΔΨm-positive parasites was determined by fluorescence microscopy after the fixation of at least 150 parasites. The diagram shows the means ± SD of data from duplicates from a representative experiment. ut, untreated. *, P < 0.002; **, P < 0.05 (determined by a Student's t test).

FIG. 5.

HDQ treatment leads to a decreased ATP level. HFFs were infected with RH strain parasites and, after 24 h, treated with 1 μM HDQ for the indicated time periods or with 1 μM oligomycin for 24 h. Intracellular parasites were released by syringe passages, and the ATP level was quantified as photons per second (CPS) in a luminescence assay. Relative ATP levels were normalized for parasite numbers. Results are expressed as means ± SD of data from duplicate wells from a representative experiment (n = 3). *, P < 0.003; **, P < 0.002; ***, P < 0.001 versus untreated control (determined by a Student's t test).

FIG. 6.

HDQ-mediated growth inhibition is not mediated by pyrimidine starvation. RH strain tachyzoites were treated with 100 nM HDQ in the presence or absence of 250 μM uracil. Duplicate samples were fixed after 24 h, and the average number of tachyzoites per vacuole was determined. At least 100 vacuoles were examined for each sample. Results are represented as means ± SD of data from a representative experiment (n = 2).

FIG. 7.

HDQ treatment upregulates transcript levels of the bradyzoite markers bag1 and enolase 1. HFFs were infected with tachyzoites and cultivated in the presence of 100 nM and 1 μM HDQ for 72 h. Enolase 1 and bag1 mRNA transcripts were determined by real-time PCR. β-Tubulin was used for normalization. The diagram shows enolase 1 and bag1 transcript levels of HDQ-treated samples relative to that of a mock-infected control (arbitrarily defined as 1), which was harvested 24 h postinfection. Results are expressed as means ± SD of data from duplicate samples from a representative experiment. *, P < 0.0001; **, P < 0.0002 versus mock control (determined by a Student's t test).

FIG. 8.

Loss of ΔΨm during bradyzoite differentiation. Bradyzoite differentiation was induced by an alkaline-pH shift (pH 8.3). At 24 h, 48 h, and 72 h postinfection, living samples were stained with DiOC₆(3) and analyzed by immunofluorescence microscopy, followed by fixation and BAG1 and Dolichos biflorus lectin staining. (A) Fluorescence images from a 72-h sample showing a DiOC₆(3)-negative/BAG1-positive/lectin-positive vacuole (top) and a vacuole that is weakly DiOC₆(3) positive (bottom). (B) Kinetics showing the decrease of DiOC₆(3)-positive vacuoles and the increase of BAG1-positive and lectin-positive vacuoles during bradyzoite differentiation. (C) Extracellular parasites were obtained after syringe passage from a 72-h bradyzoite culture and a 24-h tachyzoite culture. The diagram shows the fraction of Mitotracker-positive parasites in the BAG1-positive population (bradyzoites) in comparison to Mitotracker-positive parasites from the tachyzoite culture. More than 100 extracellular parasites were analyzed for each sample. Results are expressed as means ± standard errors of the means for data from two independent experiments.