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Eukaryotic Cell, June 2009, p. 877-887, Vol. 8, No. 6
1535-9778/09/$08.00+0     doi:10.1128/EC.00381-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

San San Lin, Uwe Groß, and Wolfgang Bohne*

Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany

Received 4 December 2008/ Accepted 2 March 2009


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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 DiOC6(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 Fo-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.


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INTRODUCTION
 
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 ({Delta}{Psi}m) (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 {Delta}{Psi}m 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 {Delta}{Psi}m and in energy metabolism in the tachyzoite stage.


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MATERIALS AND METHODS
 
T. gondii cultivation, in vitro stage conversion, and cell lines. Tachyzoites were propagated in human foreskin fibroblasts (HFFs) as previously described (30). Bradyzoite differentiation was induced by an alkaline-pH shift (34). In brief, tachyzoites were freshly released by syringe passage, and 3 x 104 parasites were inoculated onto a confluent HFF monolayer grown on a 24-well imaging plate. The pH shift medium (pH 8.3) was exchanged daily to maintain a constant pH. 143B/206 cells, which lack mitochondrial DNA (20), and the parental 143B cell line were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), 1% glutamine, sodium pyruvate (110 µg/ml), and uridine (50 µg/ml).

Chemicals. HDQ was kindly provided by W. Oettmeier and was dissolved in dimethyl sulfoxide or ethanol. Oligomycin, atovaquone, tetramethyl-p-phenylenediamine (TMPD), ascorbate, malate, succinate, glycerol-3-phosphate, dihydroorotate, oxaloacetate, uracil, and digitonin were purchased from Sigma.

Plasmids. Plasmid tub-S9-RFP/sag-CAT was constructed by replacing the FNR fragment from BglII/AvrII-digested ptub-FNR-RFP/sag CAT (36) with a BglII/AvrII S9 fragment from ptub-S9-GFP/sag-CAT (8). For generating pTet7Sag4-TgATP-β-cmyc-DHFR, the complete open reading frame (ORF) of the T. gondii ATPase β subunit (TgATP-β) (GenBank accession number DQ228960 [GenBank] ) was amplified from RH cDNA using Pfu polymerase (Promega) with the primer set consisting of primers 5'-TAATGCATAAAATGGCGTCTCCCGCACTC (NsiI) and 5'-TACCTAGGCTTTCCGCTCGCCGCTTCCTG (AvrII). The PCR fragment was cloned into pCR4.0-TOPO (Invitrogen), and the DNA was sequenced. Finally, the NsiI/AvrII fragment was subcloned into vector pTetO7Sag4-ACP-cmyc-DHFR (kindly provided by B. Striepen), thereby replacing the acyl carrier protein ORF with the ATP-β ORF. The final construct consists of the anhydrotetracycline-regulable TetO7Sag4 promoter element (25), which controls the expression of the complete TgATP-β ORF with a C-terminal myc tag, and additionally includes a pyrimethamine resistance cassette for selection (9).

Generation of transgenic parasites. The transfection of T. gondii strain RH TATi-1 (26) was carried out based on previously reported protocols (30, 35). Stable transgenic parasite lines expressing TgATP-β and S9-red fluorescent protein (RFP) were selected with 1 µM pyrimethamine and 20 µM chloramphenicol, respectively.

Detection of {Delta}{Psi}m in T. gondii. The T. gondii {Delta}{Psi}m was monitored after staining with the fluorophore Mitotracker or DiOC6(3) (3,3'-dihexyloxacarbocyanine iodine; Invitrogen). Both fluorescent dyes were dissolved in tissue-culture-grade dimethyl sulfoxide. For Mitotracker, the staining solution was prepared by adding the dye to 1% FCS-DMEM at a final concentration of 0.5 µM. For {Delta}{Psi}m detection of intracellular parasites, infected HFFs in a 24-well plate were incubated with staining solution (1 ml per well) at 37°C for 45 min. Afterwards, samples were washed with 1% FCS-DMEM, fixed with 4% paraformaldehyde-phosphate-buffered saline (PBS) for 10 min, and mounted with Moviol. In experiments using 143B/206 cells, parasites were mechanically released by syringe passage before Mitotracker staining. Freshly released parasites were harvested and immediately stained in suspension with Mitotracker solution (see above), supplemented with 25 mM HEPES (pH 7.4), for 45 min at 37°C. Parasites were fixed with 4% paraformaldehyde-PBS for 10 min after washing, and a 10-µl aliquot of the parasite suspension was air dried on a glass slide and mounted with Moviol. DiOC6(3) staining was applied for live imaging of the T. gondii {Delta}{Psi}m. The staining solution consists of 1% FCS-DMEM with a final concentration of 5 nM DiOC6(3). After incubation at 37°C for 30 min, samples were ready for real-time {Delta}{Psi}m monitoring.

Time-lapse microscopy. Live imaging of the T. gondii {Delta}{Psi}m was performed with an inverted Zeiss Axiovert 200 M microscope equipped with an XL-3 incubator and a heating unit (PeCon). Images were captured by using an AxioCam MRm camera and processed with Axiovision 4.6.3 software. All live-imaging experiments were performed on a black glass-bottomed 24-well imaging plate (Greiner Bio-One) kept at 37°C. In brief, confluent HFFs seeded onto an imaging plate were infected with 2 x 105 to 3 x 105 parasites per well. After DiOC6(3) staining, the plate was transferred to the humidified chamber, and drugs were added to the wells at the indicated concentrations.

Digitonin permeabilization. Substrate supplementation was performed on digitonin-permeabilized intracellular parasites using a modification of a previously described protocol that was established for extracellular parasites (39). Various concentrations (2 to 64 µM) of digitonin were tested on intracellular parasites, and a final concentration of 2 µM was selected, which did not cause HFF detachment and left the intensity of DiOC6(3) staining intact over a time period of 35 to 45 min. DiOC6(3)-stained intracellular parasites were digitonin permeabilized and incubated with HDQ or atovaquone, either alone or in combination with different substrates, for 15 to 25 min at the indicated concentrations. Samples were analyzed by fluorescence microscopy, and the percentage of parasites possessing a detectable {Delta}{Psi}m was determined from duplicates.

Determination of the intracellular ATP level. The parasite ATP level was determined by using the BacTiter-Glo microbial cell viability assay (Promega). Samples were prepared as follows. Infected HFFs were washed once with PBS supplemented with protease inhibitors (protease inhibitor cocktail; Roche) to remove extracellular parasites. Parasitized cells were then resuspended in 5 ml of 1% FCS-DMEM (phenol red free) supplemented with the same protease inhibitors and freshly released by syringe passage. Afterwards, a centrifugation step at 34 x g was performed in order to remove host cell debris. Parasites in the supernatant were harvested by centrifugation and resuspended in 250 µl of 1% FCS-DMEM. An aliquot of 20 µl of parasites was used for counting, and the remaining parasites were immediately frozen in liquid nitrogen for later measurement. ATP levels from each sample were measured as duplicates in a flat-bottomed 96-well plate. One hundred microliters of the parasite suspension, containing 4 x 106 parasites, was mixed thoroughly with the same volume of BacTiter-Glo reagent and incubated at room temperature for 5 min. Luminescence was detected using luminometry (Wallac 1420; Perkin-Elmer) and quantified as photon counts per second. The relative parasitic ATP levels for each sample were normalized with the numbers of parasites counted previously.

Immunofluorescence assay. Samples were first fixed with 4% paraformaldehyde-PBS for 10 min and then permeabilized with 0.25% Triton X-100-PBS for another 15 min. After blocking with 1% bovine serum albumin-PBS for 1 h, samples were incubated with an anti-myc monoclonal antibody (MAb) (clone 9E10; Sigma) (1:250), followed by a Cy2-conjugated anti-mouse immunoglobulin G (IgG) antibody (1:300; Dianova) for 1 h each. To detect bradyzoites, a bradyzoite-specific anti-BAG1 MAb (7E5) (1:500) (3) and a Cy3-conjugated anti-mouse IgG antibody (1:300; Dianova) were used instead. Lectin staining was performed with the same procedures by using a fluorescein isothiocyanate-conjugated lectin from Dolichos biflorus (1:300; Sigma).

RNA extraction and real-time PCR. Total RNA was isolated using the GenElute mammalian total RNA kit (Sigma), and reverse transcription was done using 5 µg of total RNA, oligo(dT) primer (Sigma), and Moloney murine leukemia virus reverse transcriptase (RNase H minus; Sigma) according to the manufacturer's instructions. Real-time PCR was performed using LightCycler PCR (Roche) to amplify cDNA for the mRNA transcript levels of the bradyzoite-specific genes enolase 1 and bag1 as well as β-tubulin. The primer sets used for cDNA amplification were 5'-CGAGGGGTGGCTGAAAAAGTATCC-3' and 5'-CAGCGAAGGCCCACGACAAG-3' for enolase 1, 5'-GACCGGTCGCCTCTCAACAGC-3' and 5'-CGCGCAAAATAACCGGACACT for bag1, and 5'-CGCCACGGCCGCTACCTGACT-3' and 5'-TACGCGCCTTCCTCTGCACCC-3' for β-tubulin, respectively. PCR amplification was carried out using the following parameters: 10 min at 95°C followed by 40 cycles of denaturation (95°C for 10 s), annealing (60°C for 5 s), and extension (72°C for 15 s). All samples were tested for PCR amplification efficiencies according to manufacturer's protocols and software (Roche). Serial dilutions of cDNAs were subjected to real-time PCR amplification in duplicates using the β-tubulin primer pair. Crossing points were plotted against the log of cDNA dilutions, and amplification efficiencies (E) were calculated from the slopes of the obtained lines by the following formula: E = 10-1/slope. The difference of amplification efficiencies ({Delta}E) of HDQ-treated samples and the untreated control was less than 0.05 ({Delta}EHDQ 100 nM – control = 0.019; {Delta}EHDQ 1,000 nM – control = 0.013), and crossing-point values from PCR amplification could thus be used for relative quantification. β-Tubulin mRNA levels were used for the normalization of enolase 1 and bag1 transcript levels in HDQ-treated and -untreated samples.

Protein fractionation and immunoblotting. Protein extracts were prepared at 4°C throughout. Extracellular tachyzoites (3 x 107 to 4 x 107 tachyzoites) from a stable transgenic line expressing TgATPase-β were harvested and resuspended in ice-cold PBS containing protease inhibitors. Parasites were lysed by repetitive freeze-and-thaw steps and sonicated five times for 10 s each. Subsequently, the lysates were separated after centrifugation at 13,000 x g for 45 min. The supernatant comprising the membrane-soluble fraction was collected, precipitated by trichloroacetic acid, and resuspended in 2x sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 0.09 M Tris-Cl (pH 6.8), 20% glycerol, 2% SDS, 0.02% bromophenol blue, and 0.1 M dithiothreitol. The pellet fraction was also resuspended in the same buffer. Protein fractions were resolved on an SDS-polyacrylamide gel and electroblotted onto a Hybond nitrocellulose membrane (Amersham Biosciences). Bound proteins were probed with an anti-myc MAb (1:500), followed by a secondary antibody with goat anti-mouse IgG coupled to alkaline phosphatase (1:2,000; Dianova). Reactive proteins were detected by alkaline phosphatase staining solution containing 0.05% bromo-4-chloro-3-indolyl phosphate and 0.5% nitroblue tetrazolium (Sigma) as substrates.


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RESULTS
 
HDQ treatment leads to a collapse of the {Delta}{Psi}m. The influence of the high-affinity NDH2 inhibitor HDQ (13, 22) on the {Delta}{Psi}m was investigated with the aid of the cationic fluorescent dye Mitotracker. When mock controls were stained with Mitotracker at different time points from 7 to 32 h postinfection, 75 to 80% of all intracellular parasites showed the typical intense staining of the single T. gondii mitochondrion (Fig. 1A) and were thus categorized as being {Delta}{Psi}m positive. The frequencies of {Delta}{Psi}m-positive parasites were similar in all samples and unrelated to the size of the parasitophorous vacuoles, suggesting that the majority of tachyzoites possess a {Delta}{Psi}m throughout their intracellular life span. In contrast, mitochondrial staining was absent in more than 85% of the parasites when cultures were incubated with 100 nM HDQ (Fig. 1A and B) for the last 6 h. In the remaining parasites, which were classified as being {Delta}{Psi}m positive, the intensity of the staining appeared to be less than that for the untreated controls. This suggests that HDQ treatment leads to a collapse of the {Delta}{Psi}m. Furthermore, it indicates that the susceptibility to membrane depolarization is independent from the size of the parasitophorous vacuole.


Figure 1
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  		FIG. 1. (A) HDQ treatment decreases the {Delta}{Psi}m 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 {Delta}{Psi}m-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.

Real-time monitoring of the T. gondii {Delta}{Psi}m. Mitotracker comprises a chloromethyl moiety that forms covalent bonds with the SH groups of mitochondrial membrane proteins, which allows the fixation of the stained samples before microscopic analysis. A drawback of this feature is that Mitotracker cannot be reliably used to monitor changes in the {Delta}{Psi}m in living cells, since the thiol bond formation is nonreversible. In order to determine the kinetics of an HDQ-mediated {Delta}{Psi}m collapse in intracellular T. gondii, we first searched for a {Delta}{Psi}m-sensitive dye that allows real-time imaging of the T. gondii {Delta}{Psi}m. We compared the cationic fluorophores TMRE (tetramethylrhodamine ethyl ester), JC-1 (3,3'-tetraethylbenzimidazolcarbocyanine iodine), and DiOC6(3) for their abilities to specifically stain the mitochondrion of intracellular tachyzoites with no or only weak staining of the plasma membrane. DiOC6(3) was found to be the most suitable dye for this purpose, since it resulted in an intense and specific mitochondrial staining pattern, which perfectly colocalized with a mitochondrially targeted RFP (S9-RFP) (Fig. 2A). When intracellular parasites were treated with 10 nM of the well-established complex III inhibitor atovaquone (1), the intensity of the DiOC6(3) staining decreased gradually over time, leading to a complete depolarization of the {Delta}{Psi}m within 40 min in >60% of the parasites (Fig. 2B and C). These results are consistent with previously reported observations of the mitochondrial membrane depolarization effect of atovaquone in T. gondii (39) and thus demonstrate the suitability of DiOC6(3) staining for real-time {Delta}{Psi}m imaging. The dye could be used for up to 1.5 h in real-time imaging, with a maximum of 10 to 15 exposures taken within this time. Higher numbers of exposures resulted in a significant bleaching effect, and at incubation times longer than 1.5 h, the dye showed a tendency to lose the equal distribution across the mitochondrial membrane and started to accumulate at a single location. We used DiOC6(3)-based real-time imaging in the following experiments to monitor the kinetics of HDQ-mediated {Delta}{Psi}m depolarization.


Figure 2
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  		FIG. 2. Real-time imaging of the T. gondii {Delta}{Psi}m 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 {Delta}{Psi}m 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 {Delta}{Psi}m-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.

Kinetics of HDQ-mediated {Delta}{Psi}m collapse. DiOC6(3)-based real-time imaging of the {Delta}{Psi}m for parasites treated with 10 nM, 100 nM, and 1 µM HDQ led to a dose-dependent depolarization of the T. gondii mitochondrial membrane (Fig. 2B and C). At 1 µM HDQ, the mitochondrial membrane from more than 75% of the parasites was completely depolarized within the first 5 min, demonstrating a fast mode of action of the drug (Fig. 2C). The depolarization kinetics of 10 nM HDQ were similar to those of 10 nM atovaquone. A combination of 10 nM HDQ with 10 nM atovaquone resulted in a significantly faster collapse of the {Delta}{Psi}m than treatment with 10 nM of the individual drugs, which is in agreement with a synergistic mode of action between HDQ and atovaquone (31).

Substrates for ubiquinone-reducing enzymes lead to {Delta}{Psi}m stabilization. Aside from the two type II NADH dehydrogenases, T. gondii possesses a further four enzymes that can feed electrons into the ubiquinol pool, namely, succinate dehydrogenase, malate:quinone oxidoreductase, dihydroorotate dehydrogenase (DHODH), and glycerol-3-phosphate dehydrogenase. We investigated whether an excess of substrates for these enzymes could compensate for an HDQ-mediated depolarization of the {Delta}{Psi}m. Cells were treated for this purpose with 2 µM digitonin, a concentration which was shown previously to selectively permeabilize the parasite's plasma membrane for metabolites without disturbing the function of the respiratory chain (39). The {Delta}{Psi}m of living, intracellular parasites was monitored by DiOC6(3) staining. In drug-untreated controls, more than 80% of the parasites displayed a strong {Delta}{Psi}m, confirming that the digitonin treatment itself did not affect the {Delta}{Psi}m (Fig. 3). Treatment with TMPD-ascorbate, a combination which is commonly used to feed electrons into complex IV, led to a strongly attenuated {Delta}{Psi}m depolarization after HDQ treatment, indicating that HDQ inhibits the ETC upstream of complex IV. The addition of dihydroorotate, glycerol-3-phosphate, malate, or succinate did not prevent an atovaquone-mediated {Delta}{Psi}m collapse. This was the expected result, since atovaquone, as a complex III inhibitor, blocks the ETC downstream of ubiquinone reduction. However, each of the four substrates significantly increased the number of {Delta}{Psi}m-positive parasites in the presence of HDQ (Fig. 3). The highest number of {Delta}{Psi}m-positive parasites in HDQ-treated cultures was achieved when all four substrates were added simultaneously. Oxalacetate was used as a control and did not result in an increased frequency of {Delta}{Psi}m-positive parasites. Together, these results suggest that HDQ possesses a different mode of action compared to that of atovaquone and inhibits the ETC upstream of complex III at the level of ubiquinone reduction.


Figure 3
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  		FIG. 3. Substrate supplementation in permeabilized parasites partly decreases HDQ-mediated {Delta}{Psi}m 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 {Delta}{Psi}m-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).

Oligomycin-mediated inhibition of the T. gondii FoF1-ATPase attenuates HDQ-mediated {Delta}{Psi}m depolarization. We next investigated whether the {Delta}{Psi}m can be stabilized in HDQ-treated parasites by an inhibition of the putative T. gondii FoF1-ATPase. The inner mitochondrial membrane is impermeable to protons, and the only possibility for protons to reenter the mitochondrial matrix is through the Fo proton channel, which can be inhibited by oligomycin. The T. gondii genome contains all genes for the five parts ({alpha}, β, {gamma}, {delta}, and {varepsilon}) that form the F1 subunit. However, the parasite appears to possess an unusual Fo subunit, since from the three proteins (Fo-a, Fo-b, and Fo-c) that typically form the Fo subunit, obvious homologues for Fo-a and Fo-b are lacking (23). This indicates either a high degree of divergence in the lacking parts or an unusual composition of the Fo subunit. In Plasmodium, which is lacking all three Fo-forming proteins, a matrix localization of the F1 subunit was previously proposed, which implies that the proton gradient cannot be used for ATP synthesis (29). In order to discriminate between a matrix and a membrane association of the F1 subunit, we examined the localization of the F1-ATPase in parasites, which expressed an epitope-tagged version of ATPase-β (TgATP-β), which is a part of the F1-ATPase. Myc-tagged TgATP-β of stably transfected parasites was targeted to the mitochondrion, as shown by the colocalization with the Mitotracker signal (Fig. 4A). After fractionation of the T. gondii lysate, ATPase-β was found exclusively in the membrane fraction and was absent in the soluble fraction, suggesting that T. gondii possesses a typical, membrane-associated mitochondrial F1-ATPase (Fig. 4B).


Figure 4
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  		FIG. 4. Inhibition of membrane-associated T. gondii FoF1-ATPase attenuates HDQ-mediated {Delta}{Psi}m 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 {Delta}{Psi}m-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).

The effect of oligomycin-mediated FoF1-ATPase inhibition was further investigated by using 143B/260 cells as host cells for T. gondii. This cell line lacks a functional mitochondrial respiratory chain (20), which excludes the possibility that oligomycin has an indirect effect on T. gondii via an inhibition of host cell ATP synthesis. The fraction of parasites with positive Mitotracker staining was determined using 143B/260 host cells after HDQ treatment with and without the addition of oligomycin. Oligomycin treatment resulted in a strong increase in levels of {Delta}{Psi}m-positive parasites (Fig. 4C), suggesting that the HDQ-mediated {Delta}{Psi}m depolarization can be attenuated by preventing protons from reentering the mitochondrial matrix.

HDQ treatment leads to a decreased ATP level. We further examined the influence of HDQ-mediated {Delta}{Psi}m collapse on the parasitic ATP level. Infected cultures were incubated with 1 µM HDQ, and intracellular parasites were mechanically released from host cells by syringe passage at 1, 3, 8, and 24 h after the addition of HDQ. The ATP level of the harvested parasites was determined using a luminescence assay, and the obtained values were normalized for parasite numbers. An oligomycin-treated sample was included in order to quantify ATP levels in parasites in which FoF1-ATPase activity was inhibited. The kinetics revealed that HDQ leads to a gradual decrease of the parasite's total ATP level, resulting in a ~30% reduction after 1 h and a ~70% reduction after 24 h (Fig. 5). A 70% decrease in the ATP level was also observed after the complete inhibition of FoF1-ATPase activity using 1 µM oligomycin.


Figure 5
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  		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).

HDQ growth inhibition is not mediated by pyrimidine starvation. HDQ possesses structural similarities to ubiquinol and is believed to interact with the ubiquinol binding site of type II NADH dehydrogenases (13). One of the enzymes also possessing a ubiquinol binding site is DHODH, which catalyzes the fourth step in de novo pyrimidine biosynthesis. Since P. falciparum DHODH was recently described to be inhibited by HDQ (10), we examined whether HDQ exerts its growth-inhibitory effect on T. gondii by pyrimidine starvation. T. gondii possesses a pyrimidine salvage pathway, and parasites deficient in de novo pyrimidine synthesis can be rescued with high concentrations of uracil, which is converted by the parasitic uracil-phosphoribosyltransferase to UMP (19). We supplemented the culture medium with 250 µM uracil and determined the T. gondii growth rate in the presence of 100 nM HDQ. Uracil supplementation did not lead to an increased growth rate (Fig. 6), suggesting that the underlying mechanism of HDQ growth inhibition is not due to pyrimidine starvation.


Figure 6
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  		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).

HDQ induces bradyzoite differentiation. HDQ was shown to effectively inhibit parasite replication (31). Since a reduction in the level of parasite replication in T. gondii is often associated with stage differentiation (4), we investigated whether HDQ treatment leads to bradyzoite differentiation. HFFs were infected with tachyzoites and cultivated in the presence of 100 nM and 1 µM HDQ for 72 h. Subsequently, the transcript levels of the bradyzoite marker genes bag1 (5) and enolase 1 (11) were determined by real-time PCR, in comparison to drug-untreated controls. Both genes were upregulated between six- and ninefold after HDQ treatment (Fig. 7), indicating that HDQ leads to a moderate induction of bradyzoite differentiation.


Figure 7
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  		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).

The frequency of {Delta}{Psi}m-positive parasites decreases during bradyzoite differentiation. There are several indications that the energy metabolism of bradyzoites is different from that of tachyzoites (12, 41), and it was thus of interest to compare the frequencies of {Delta}{Psi}m-positive parasites in both stages. Bradyzoite differentiation was induced by an alkaline-pH shift, and the percentage of {Delta}{Psi}m-positive parasites was determined after DiOC6(3) staining in living cultures at 24 h, 48 h, and 72 h postinfection. The rate of bradyzoite differentiation was determined using the same samples after fixation and staining with a bradyzoite-specific anti-BAG1 antibody, which detects a cytosolic small heat shock protein, and with a fluorescein isothiocyanate-conjugated Dolichos biflorus lectin, which detects a carbohydrate structure on the emerging cyst wall (Fig. 8A). The fraction of {Delta}{Psi}m-positive parasites gradually decreased from ~85% after 24 h to 20% after 72 h, while the expression of the bradyzoite markers increased to 70 to 80% (Fig. 8B). This suggests that a fraction of parasites lost the {Delta}{Psi}m during bradyzoite differentiation. A putative concern was that the emerging cyst wall acts as a diffusion barrier and prevents the access of DiOC6(3) to the bradyzoites. To exclude this possibility, we verified the results using freshly harvested extracellular bradyzoites, which were released from their parasitophorous vacuoles and the emerging cyst wall by extensive syringe passage. Mitotracker staining was applied for the released parasites, followed by fixation, permeabilization, and BAG1 staining. The frequency of Mitotracker-positive parasites in the BAG1-positive population was less than 10%, compared to ~80% Mitotracker-positive parasites in freshly released extracellular tachyzoites (Fig. 8C). These results are in agreement with the data obtained from intracellular parasites and confirm the strong decrease in levels of {Delta}{Psi}m-positive parasites during bradyzoite differentiation.


Figure 8
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  		FIG. 8. Loss of {Delta}{Psi}m 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.


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DISCUSSION
 
We demonstrate in this study that the treatment of intracellular tachyzoites with HDQ, a quinolone-like compound that was previously shown to inhibit TgNDH2-I (22), leads to a fast, dose-dependent collapse of the {Delta}{Psi}m and subsequently to a decrease in the intracellular ATP level. The mode of action of HDQ in T. gondii is thus an inhibition of oxidative phosphorylation. The observed synergism of 10 nM HDQ in combination with 10 nM atovaquone on {Delta}{Psi}m depolarization is in agreement with the synergism of these drugs to inhibit parasite replication in vitro (31). The addition of succinate, dihydroorotate, glycerol-3-phosphate, or malate to digitonin-permeabilized cells stabilized the {Delta}{Psi}m in the presence of HDQ, whereas these substrates did not influence atovaquone-mediated depolarization. This observation clearly indicates that HDQ acts as an ETC inhibitor upstream of the atovaquone target, which is complex III, at the level of ubiquinone reduction. The attenuation of HDQ-mediated {Delta}{Psi}m depolarization in the presence of high substrate concentrations of ubiquinone-reducing enzymes is in agreement with HDQ acting as an NDH2 inhibitor and a replenishment of the ubiquinol pool by an increased level of activity of the corresponding enzymes succinate dehydrogenase, DHODH, glycerol-3-phosphate dehydrogenase, and malate:quinone oxidoreductase. Since physiological substrate concentrations in non-digitonin-treated cells are not sufficient to compensate for the HDQ-mediated {Delta}{Psi}m depolarization, a major role of NDH2 activity in providing the ETC with reduction equivalents can be assumed under the assumption that HDQ is not affecting any other targets of the ETC. Although we have no indications for potential other targets of HDQ in T. gondii, we cannot completely rule out the possibility that HDQ also exerts an inhibitory effect on one or more of the above-mentioned ubiquinone-reducing enzymes. However, we excluded the possibility that pyrimidine starvation is the mode of action by which HDQ inhibits T. gondii replication. A recent study reported that HDQ inhibits DHODH of P. falciparum (10). This ubiquinone-dependent enzyme catalyzes the dehydrogenation of dihydroorotate to orotate, an essential step for de novo pyrimidine biosynthesis. In contrast to Plasmodium, T. gondii possesses a pyrimidine salvage pathway, and parasites deficient in de novo pyrimidine synthesis can be rescued with high uracil concentrations (19). Since uracil supplementation did not rescue parasite replication in HDQ-treated cultures, we could exclude that pyrimidine starvation is the major mode of inhibition of HDQ in T. gondii.

FoF1-ATPases use the proton motive force across the inner mitochondrial membrane for coupling proton translocation through a membrane-bound, oligomycin-sensitive Fo subunit with ATP synthesis at the F1 subunit. The Fo subunit is typically composed of three proteins (Fo-a, Fo-b, and Fo-c); however, the T. gondii genome has no obvious homologues for the Fo-a and Fo-b proteins (23). It was thus unclear whether the T. gondii F1 subunit is indeed associated with a putative membrane-bound Fo subunit or if this enzyme is localized in the mitochondrial matrix, as proposed previously for Plasmodium (29), which is lacking all three parts of the Fo subunit. The latter would imply that the proton motive force cannot be used for ATP synthesis. We showed by subcellular fractionation that the F1 subunit is associated exclusively with the membrane fraction, which suggests an interaction of F1 with a membrane-associated Fo or Fo-like subunit. A conventional function of the T. gondii FoF1-ATPase in coupling the proton gradient with ATP synthesis is consistent with our observation that (i) an HDQ-mediated depolarization of the inner mitochondrial membrane leads to a ~30% reduction of the ATP level within 1 h and to a ~70% reduction within 24 h, (ii) treatment with the Fo subunit inhibitor oligomycin leads to a ~70% reduction of the ATP level, and (iii) oligomycin leads to a stabilization of the {Delta}{Psi}m in the presence of HDQ. These results are in agreement with data from previous biochemical analyses in which the level of O2 consumption of digitonin-treated extracellular T. gondii was shown to be increased in the presence of ADP and decreased in the presence of the Fo subunit inhibitor oligomycin (39).

The relative contribution of oxidative phosphorylation to total ATP synthesis is still a matter of debate for T. gondii, as it is for other apicomplexan parasites (24, 33). Our studies suggest that oxidative phosphorylation is indispensable for sufficient ATP generation in the growing-tachyzoite stage and that other ATP-generating pathways such as glycolysis cannot fully compensate for its loss. The HDQ-mediated depolarization of the {Delta}{Psi}m occurs within minutes, while the onset of the ATP decrease started with a delay of ~30 min. It is conceivable that a reserve energy system of limited capacity contributes to a stable ATP amount within the first minutes after the inhibition of oxidative phosphorylation. A likely candidate for such an energy buffer system is the adenylate kinase reaction, which converts two ADP molecules into ATP and AMP in a reversible reaction. A common response to the inhibition of oxidative phosphorylation, which might also occur in T. gondii, is an increased metabolic flux through other energy-generating pathways, like glycolysis. High substrate concentrations at the beginning of the inhibition process, for example, of glucose-6-phosphate, might also contribute to a timely, limited stabilization of the ATP level until these resources are reduced in concentration.

The fraction of parasites with a detectable {Delta}{Psi}m is not constant throughout the life cycle but is strongly decreased during tachyzoite-to-bradyzoite conversion. This is in agreement with the concept that T. gondii adapts its metabolism during the transition from tachyzoites to long-term persistent bradyzoites, which are believed to possess a reduced metabolism (4, 12, 41). The close link between energy metabolism and stage conversion is furthermore supported by our observation that long-term treatment with HDQ for 3 days leads to an upregulation of mRNA transcripts for bradyzoite markers. This is in agreement with previously reported observations that inhibitors of the respiratory chain and of oxidative phosphorylation lead to an induction of bradyzoite differentiation (4, 37). The parasite appears to respond in a situation of energy starvation with a differentiation into the dormant stage.


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ACKNOWLEDGMENTS
 
HDQ was kindly provided by W. Oettmeier (University of Bochum).

This study has been supported by a grant from the Deutsche Forschungsgemeinschaft to W.B. (BO 1557/3-1). S.S.L. is supported by a Croucher overseas scholarship award.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany. Phone: 49-551-395869. Fax: 49-551-395861. E-mail: wbohne{at}gwdg.de Back

{triangledown} Published ahead of print on 13 March 2009. Back


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Eukaryotic Cell, June 2009, p. 877-887, Vol. 8, No. 6
1535-9778/09/$08.00+0     doi:10.1128/EC.00381-08
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