• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. Mar 2011; 55(3): 1045–1052.
Published online Jan 3, 2011. doi:  10.1128/AAC.01545-10
PMCID: PMC3067095

Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance in Leishmania major[down-pointing small open triangle]

Abstract

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.

Treatment for leishmaniasis currently relies on a reduced arsenal of drugs, including pentavalent antimonials (which cannot be given in areas where drug resistance is endemic), amphotericin B deoxycholate, lipid formulations of amphotericin B, miltefosine, and paromomycin, all of which have drawbacks in terms of toxicity, efficacy, price, and inconvenient treatment schedules (6, 17). To increase the therapeutic life span of these drugs and delay the emergence of resistance, the World Health Organization has recommended combination therapy as a strategy to be implemented in clinical trials. Of the new drugs under development, 8-aminoquinolines such as sitamaquine (WR6026; GlaxoSmithKline), which is currently in phase 2b clinical trials (12, 34), represent a promising new oral leishmanicidal treatment. Although the mechanism of action of sitamaquine is still unknown, it has been reported that acidocalcisomes play a key role in the accumulation of sitamaquine, although they do not determine the leishmanicidal potency of the drug (14). Other 8-aminoquinolines chemically related to primaquine have been synthesized and evaluated for their antiparasitic activity (1, 25). Thus, tafenoquine (TFQ), formerly known as WR238605, is a primaquine analogue which is being developed jointly by the Walter Reed Army Institute of Research and GlaxoSmithKline pharmaceuticals for the treatment and prevention of relapsing malaria (31). Phase I, II, and III clinical studies with this drug have shown that TFQ is a safe, well-tolerated, and highly effective oral chemoprophylactic agent for the treatment of plasmodial infections (8, 24, 30). Recently, we have proposed that TFQ could be used as a new leishmanicidal drug (33) and have determined that TFQ targets Leishmania mitochondria by specifically inhibiting mitochondrial cytochrome c reductase, thus leading to a final apoptosis-like process (3). However, to ensure the future long life of this promising leishmanicidal drug, it is important to determine how easy it is to induce TFQ resistance experimentally, as this information can then be extrapolated to the possible emergence of drug resistance in the field. The mechanism of resistance to other aminoquinoline derivatives, such as the 4-aminoquinoline derivative chloroquine, in Plasmodium, for example, has been associated with a reduction in drug accumulation (13, 27).

In the present study, we have determined the mechanism of resistance to TFQ in Leishmania parasites and have found that TFQ resistance is unstable. TFQ accumulation was lower in resistant parasites than in sensitive parasites, although reduced drug accumulation was found not to be a mechanism of resistance, as it was present in revertant (i.e., nonresistant) lines. However, the mechanism of TFQ resistance does appear to be linked to increased ATP synthesis from glycolysis.

MATERIALS AND METHODS

Chemical compounds.

TFQ [2-methoxy-4-methyl-5-(3-trifluoromethylphenoxy)primaquine succinate], sitamaquine [N,N-diethyl-N′-(6-methoxy-4-methylquinolin-8-yl)hexane-1,6-diamine] dihydrochloride and [benzene ring-U-14C]sitamaquine ([14C]sitamaquine; 2.07 GBq/mmol) were provided by GlaxoSmithKline (Greenford, United Kingdom). [14C]Glucose (11.8 GBq/mmol) was purchased from PerkinElmer. Amplex Red, LysoTracker Green DND-26, LysoTracker Red DND-99, BCECF-AM [2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester], and BCECF (free acid) were purchased from Invitrogen. Dicyclohexylcarbodiimide (DCCD), trivalent antimony, pentamidine, paromomycin, amphotericin B, ketoconazole, chloroquine, quinine, mefloquine, primaquine, glucose, sodium azide, ammonium chloride, nigericin sodium salt, 2-deoxy-d-glucose, DAPI (4′,6-diamidino-2-phenylindole dilactate), phosphoenolpyruvate, propidium iodide, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], phenylmethanesulfonyl fluoride (PMSF), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), horseradish peroxidase (HRP), pyruvate oxidase, and sodium pyruvate were purchased from Sigma-Aldrich (St. Louis, MO). Lactate dehydrogenase was purchased from Roche Applied Science, and miltefosine was purchased from Æterna Zentaris (Frankfurt, Germany). All chemicals were of the highest quality available.

Leishmania culture conditions.

Promastigotes of Leishmania major (MHOM/JL/80/Friedlin) and derivative lines used in this study were cultured at 28°C in RPMI 1640 modified medium (Invitrogen, Carlsbad, CA) supplemented with 20% heat-inactivated fetal bovine serum (iFBS; Invitrogen), as described previously (10). All parasite lines were collected from culture by centrifugation after 48 h of growth and washed in phosphate-buffered saline (PBS; 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 130 mM NaCl, and 2.6 mM KCl adjusted to pH 7). The final parasite concentration was determined using a Coulter Z1 counter.

Generation of a TFQ-resistant L. major line.

A TFQ-resistant L. major line was obtained by following a previously described stepwise selection process (4, 23) with a starting concentration of 2.5 μM TFQ increasing to 4 μM TFQ over 10 weeks. The TFQ-resistant (R4) line was maintained in the continuous presence of 4 μM TFQ. To determine the stability of the resistant phenotype, the R4 line was grown in a drug-free medium for 1 month (revertant line; revR4). The sensitivity of wild-type (WT), R4 and revR4 L. major promastigotes to TFQ and the cross-resistance profile of the R4 line to different compounds were determined after incubation for 72 h at 28°C in the presence of increasing concentrations of the drug. The concentration of TFQ required to inhibit parasite growth by 50% (EC50), and the resistance indices (ratio of EC50s for resistant and WT parasites) were calculated using an MTT colorimetric assay, as described previously (11).

TFQ sensitivity in intracellular amastigotes of Leishmania.

Late-stage promastigotes from the WT and R4 lines were used to infect mouse peritoneal macrophages from BALB/c mice (Charles River, Ltd.) at a macrophage/parasite ratio of 1:10, as described previously (14). After infection for 6 h, extracellular parasites were removed by washing with serum-free medium. Infected macrophage cultures were maintained at 37°C with 5% CO2 at different TFQ concentrations in RPMI 1640 medium plus 10% iFBS, as described previously (14). After 72 h, macrophages were fixed for 20 min at 4°C with 2.5% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and treated with RNase A (1 mg/ml) for 30 min. Intracellular parasites were detected by nuclear staining with 5 μg/ml propidium iodide and Prolong Gold antifade reagent without DAPI (Invitrogen).

TFQ uptake and efflux.

L. major promastigotes (2 × 107 per ml) were incubated with 5 μM TFQ for 15 min at 28°C in culture medium and then washed twice with PBS and lysed by the addition of 10% SDS, pH 4. Sample fluorescence (in the range of 360 to 460 nm) was then measured using an Aminco Bowman series 2 spectrometer upon excitation at 340 nm. The time course uptake of TFQ at 28°C was determined at different time intervals (1, 3, 5, 8, 10, and 15 min). To determine TFQ efflux, WT and R4 parasites (2 × 107 per ml) were incubated with 2 and 2.5 μM TFQ, respectively, for 1 h in culture medium at 28°C to allow for a similar labeling in the two lines. The parasites were then washed with PBS and resuspended in culture medium at 28°C, and the fluorescence retained was measured at different time points (0, 15, 30, 60, and 90 min).

Accumulation of sitamaquine.

Sitamaquine accumulation was determined as described previously (14). Briefly, promastigotes were incubated at 28°C with 0.5 μM [14C]sitamaquine for 15 min and then washed with PBS containing 100 μM nonradioactive sitamaquine. The cell-associated radioactivity was measured by liquid scintillation counting, and the protein concentration was determined using a Bradford kit (Bio-Rad).

Microscopy analysis.

Promastigotes (107 per ml) were incubated with 5 μM TFQ for 15 min in culture medium. After being washed twice with PBS, the pellet was resuspended in PBS and analyzed by fluorescence microscopy (Zeiss). Acidocalcisome accumulation of the acidotropic dye LysoTracker Red DND-99 was determined by confocal microscopy analysis, as described previously (14). Briefly, promastigotes were incubated with 75 nM LysoTracker Red DND-99 in HBS buffer (21 mM HEPES, 0.7 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, and 6 mM glucose [pH 7]), and the parasites were then washed with PBS and analyzed with a confocal microscope (TCS SP5 model; Leica) equipped with a He/Ne laser (633 nm) and coupled to an MRC1024 model confocal scanning laser equipment.

pHi measurement.

The intracellular pH (pHi) of L. major lines was determined fluorimetrically using a BCECF-AM probe, as described previously (28). Briefly, promastigotes (2 × 107 per ml) were resuspended in standard buffer (136.89 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, 8.46 mM Na2HPO4, 11.1 mM glucose, 1 mM CaCl2, 0.8 mM MgSO4, and 20 mM HEPES [pH 7.4]). After incubation for 30 min at 28°C in the presence of BCECF-AM (5 μg/ml), the parasites were washed twice. The fluorescence ratios (excitation ratio, 490 nm/440 nm; emission, 535 nm) were monitored continuously using an Aminco Bowman series 2 spectrometer. For calibration curves, the BCECF ratio fluorescence as a function of pH was obtained using a parasite suspension incubated with BCECF-AM and treated with the ionophore nigericin (5 μg/ml). Different pH values were obtained by the addition of 1 M MES (morpholineethanesulfonic acid; pH 5.0) or 1.5 M Tris-ClH (pH 8.8) followed by measurement of the pH. The intracellular alkalinization of parasites was obtained after pretreatment of L. major lines at 28°C with 20 mM NH4Cl for 1 min, followed by incubation with 5 μM TFQ in the presence of NH4Cl for 15 min. For the studies involving parasite acidification, the pHi was lowered using the NH4Cl prepulse technique, as described previously (15). Thus, promastigotes (2 × 107) were resuspended in 50 μl of standard buffer (described above) containing 40 mM NH4Cl at 28°C for 15 min. The parasites were then centrifuged and resuspended in standard buffer with or without the H+-ATPase inhibitor DCCD and in chloride-free buffer (135 mM sodium gluconate, 5 mM potassium gluconate, 5 mM glucose, 1 mM calcium gluconate, 1 mM MgSO4, and 10 mM HEPES-Tris [pH 7.4]). The rate of recovery from acidification was determined from the slope of the initial 100 s of recovery, and the final pHi was determined after 10 min. To determine the acidification produced by TFQ and sodium azide, BCECF-loaded promastigotes (2 × 107 per ml) were incubated in culture medium with 5 μM TFQ or 20 mM sodium azide for different times (1, 3, 5, 10, and 30 min).

Proton efflux measurements for Leishmania parasites.

Extracellular pH, which reflects the proton efflux activity of parasites, was determined as described previously (15). Thus, parasites (1 × 108) were washed and resuspended in 2 ml of weakly buffered (0.1 mM HEPES-Tris; pH 7.4) standard buffer containing 0.38 μM BCECF (free acid). The fluorescence excitation ratio (490 nm/440 nm; emission, 535 nm) was then recorded and translated into nmol of H+ released/min on the basis of ratios obtained at various extracellular pHs by the addition of known HCl equivalents.

ATP measurements for L. major lines.

ATP was measured using a CellTiter-Glo luminescent assay (Promega), which generates a luminescent signal proportional to the amount of ATP present. Promastigotes (4 × 106 per ml) were incubated in culture medium at different TFQ concentrations (1, 2, 5, 10, and 20 μM) for 60 min. For glycolytic and mitochondrial ATP determination, parasites were incubated separately in HBS buffer plus 20 mM sodium azide to inhibit mitochondrial oxidative ATP generation, and glucose-free HBS buffer plus 5 mM 2-deoxy-d-glucose and 5 mM sodium pyruvate to inhibit glycolytic ATP generation, for 1 h at 28°C. For the study of ATP levels at different pHi values, promastigotes (2 × 107 per ml) were incubated in distinct buffers to obtain a different pHi, as described previously (15). The buffers used were regular buffer (135 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgSO4 and 10 mM HEPES-Tris for pH 7.4 or 10 mM MES-Tris for pH 5.5), regular buffer containing 25 mM HCO3, sodium-free buffer (135 mM choline Cl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgSO4 and 10 mM HEPES-Tris [pH 7.4]) and chloride-free buffer (described above). The pHi of parasites was measured using BCECF-AM as the pHi fluorescent probe, as described above. A 25-μl aliquot of parasites was then transferred to a 96-well plate, mixed with the same volume of CellTiter-Glo, and incubated in the dark for 10 min, and the bioluminescence was measured using an Infinite F200 microplate reader (Tecan Austria GmbH, Austria).

Determination of intracellular pyruvate levels in Leishmania lines.

Pyruvate was extracted as described by Zhu et al. (35). Briefly, 5 × 108 parasites were incubated in HBS buffer or in glucose-free HBS buffer plus 5 mM 2-deoxy-d-glucose for 1 h at 28°C. The parasites were then collected and resuspended in 0.1 ml of ice-cold 0.25 M HClO4 and incubated on ice for 5 min. The resulting mixture was neutralized with 2.8 μl of 5 M K2CO3 (pH ≈6.5). The supernatant was collected after centrifugation at 10,000 × g for 5 min. Pyruvate was measured using a fluorimetric assay (35) based on the oxidation of pyruvate by pyruvate oxidase. The hydrogen peroxide generated reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the red fluorescent product resorufin. This fluorescence is proportional to the initial pyruvate concentration in the solution. Briefly, 20 μl of intracellular pyruvate extract was pipetted into a 96-well white plate, and 180 μl of reaction solution (final concentration, 100 mM potassium phosphate with 1 mM EDTA [pH 6.7], 1 mM MgCl2, 10 μM FAD, 0.2 mM TPP, 0.2 U/ml pyruvate oxidase, 50 μM Amplex Red, and 0.2 U/ml HRP) was then added. Fluorescence at 590 nm was measured upon excitation at 535 nm using a microplate reader (Infinite F200; Tecan Austria GmbH, Austria).

Determination of pyruvate kinase activity in Leishmania lines.

Pyruvate kinase activity was determined as described previously (21). Briefly, parasites (1 × 108) were washed twice in PBS, resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.4) plus a protease inhibitor cocktail (Sigma-Aldrich) for 10 min at 4°C, and then disrupted by nitrogen cavitation (90 bar, 30 min, 4°C). Cell debris was removed by centrifugation at 800 × g and 4°C for 10 min, and the supernatant was then centrifuged at 100,000 × g and 4°C for 60 min. Pyruvate kinase activity was determined with a lactate dehydrogenase-coupled assay system by measuring the decrease of NADH absorbance at 340 nm. The enzymatic assay was performed at 25°C in 1 ml of reaction mixture containing 50 mM triethanolamine buffer (pH 7.2), 2.5 mM phosphoenolpyruvate, 2 mM ADP, 6 mM MgSO4, 50 mM KCl, 0.42 mM NADH, and 6.25 μg lactate dehydrogenase. The reaction was initiated by the addition of 50 μg of protein from the supernatant from the centrifugation performed at 100,000 × g.

Glucose uptake assay for Leishmania lines.

The accumulation of glucose was determined after incubation of 2 × 107 promastigotes with 0.15 μCi/ml [14C]glucose for 10 min at 28°C in glucose-free HBS buffer. The parasites were subsequently washed with PBS containing 6 mM nonradioactive glucose at 4°C and then with PBS alone. The cell-associated radioactivity was measured by liquid scintillation counting, and the protein concentration was determined using a Bradford kit (Bio-Rad).

Statistical analysis.

Statistical significance was calculated by using Student's t test. Differences were considered significant at a P value of <0.05.

RESULTS

Generation of a TFQ-resistant L. major line.

A TFQ-resistant L. major line was selected in vitro by a stepwise adaptation process up to a maximum of 4 μM TFQ. Subsequent attempts to increase the level of TFQ resistance were unsuccessful. This TFQ-resistant line (R4) has an EC50 of 5.45 ± 0.35 μM, a 2.5-fold-higher concentration than for the WT line (Fig. (Fig.11 and Table Table1).1). The R4 parasites showed a growth rate similar to that of the WT line. Additionally, no morphological differences were observed between the two lines, as determined by light microscopy and flow cytometry analyses (data not shown). The resistance phenotype was unstable in drug-free medium for up to 4 weeks; the revertant line (revR4) presented a sensitivity (2.26 ± 0.02 μM) that was similar to that of the WT parasites (Fig. (Fig.1).1). Additionally, we found that TFQ resistance in the promastigote forms was maintained in intracellular amastigotes obtained after infection of mouse peritoneal macrophages with WT and R4 promastigotes (EC50s of 0.38 ± 0.03 and 0.95 ± 0.02 μM for intracellular WT and R4 amastigotes, respectively) and that R4 amastigotes were 2.5-fold more resistant, similar to the value obtained for R4 promastigotes. Additionally, the R4 line profile showed significant cross-resistance to the 8-aminoquinolines sitamaquine (2-fold more resistant) and primaquine (1.9-fold; Table Table1);1); no cross-resistance to the 4-aminoquinolines chloroquine and mefloquine and the quinoline derivative quinine was observed. As can be seen from Table Table1,1, the R4 parasites did not show any significant cross-resistance to other antileishmanial drugs, such as trivalent antimony, pentamidine, miltefosine, paromomycin, and ketoconazole.

FIG. 1.
TFQ sensitivity of L. major promastigotes. L. major WT (diamonds), R4 (squares), and revR4 (triangles) lines were assayed for TFQ sensitivity by determination of the percent cell viability using an MTT-based assay, as described in Materials and Methods. ...
TABLE 1.
Drug sensitivity profile for L. major promastigote linesa

TFQ uptake and accumulation in acidic vesicles.

To determine whether TFQ resistance in R4 parasites was associated with a failure to accumulate the drug, TFQ uptake experiments were therefore carried out using spectrofluorometric techniques. The time course uptake of TFQ in WT, R4, and revR4 lines showed that TFQ uptake became saturated after 5 min (data not shown), with a 24% lower uptake in R4 parasites with respect to their WT counterparts and intermediate values for the revR4 line (data not shown). Additionally, no significant differences in TFQ efflux between the R4 and WT parasites were observed (data not shown). A decrease in R4 and revR4 drug uptake was also observed for [14C]sitamaquine, which present a 55% lower uptake in R4 parasites than in WT parasites, and the revR4 line presents accumulation values of 25% (data not shown). Since TFQ and sitamaquine accumulate in acidic vesicles, such as acidocalcisomes (3, 14), the differences in the size of such organelles could determine the TFQ accumulation. In fact, with the acidic organelles in WT parasites being larger than those in the R4 and revR4 lines, as can be seen from the significantly lower accumulation of LysoTracker Green (data not shown), a fluorescent acidotropic probe was used to label acidic organelles (mainly acidocalcisomes) in Leishmania (18).

An increase in pHi reduces TFQ accumulation.

The accumulation of weakly basic aminoquinolines, such as amodiaquine, chloroquine, and more recently, sitamaquine, seems to be pH dependent (9, 14). Initially, we determined the pHi of Leishmania lines using BCECF as a fluorescent-dependent pH indicator (28). Under physiological culture conditions, WT promastigotes maintained a steady-state pHi of 6.75 ± 0.01 (n = 5) (Fig. (Fig.2),2), a value similar to that reported by other authors (28), whereas R4 parasites presented a pHi of 7.21 ± 0.02 (n = 5) (Fig. (Fig.2),2), which is 0.46 pH units higher than that observed for WT parasites. Interestingly, a similar pHi alkalinization was observed in revR4 parasites, which presented a pHi of 7.11 ± 0.01 (Fig. (Fig.2),2), 0.36 units higher than that for WT parasites. These findings suggest that an increased pHi could be associated, in part, with decreased TFQ accumulation in R4 parasites. To validate this hypothesis, we induced an intracellular alkalinization of parasites with NH4Cl and then incubated the parasites with TFQ. The pHi obtained for WT parasites pretreated with NH4Cl increased by 0.94 units (n = 4) and was associated with a significant reduction in TFQ accumulation (close to 35%) with respect to the control parasites (Table (Table2).2). Similar experiments using R4 and revR4 parasites led to a moderate increase in the pHi values (0.24 and 0.30 units, respectively), both of which were associated with a lower, but nevertheless significant, decrease in TFQ accumulation (4% and 11.7% for R4 and revR4, respectively; Table Table22).

FIG. 2.
Determination of intracellular pH (pHi) in L. major lines. Leishmania WT promastigotes loaded with BCECF-AM (5 μg/ml, 30 min) were exposed to a KCl/HEPES-nigericin (NIG)-containing solution (pH 7.1). The traces represent the fluorescence signal ...
TABLE 2.
pHi dependence of tafenoquine accumulation in Leishmania promastigotesa

Activation of a plasma membrane P-type H+-ATPase in R4 parasites.

One proposed mechanism for pHi regulation in Leishmania promastigotes involves a plasma membrane P-type H+-ATPase (15) identified for other unicellular organisms (15, 28). In light of the possibility that the increased pHi observed with the R4 and revR4 Leishmania lines could be due to an increased P-type H+-ATPase activity, we determined the proton efflux activity as described previously (15). The fluorescence ratios obtained (excitation at 505/440 nm and emission detected at 530 nm) were transformed into the amount of H+ released min−1. The results confirmed that the proton efflux in R4 parasites was significantly higher, with a value of 24.58 ± 0.05 nmol H+/min per 108 cells (7.65 ± 0.44 nmol H+/min per 108 WT parasites). The R4 parasites therefore presented a higher proton efflux activity (3.21-fold) than WT parasites as a mechanism of pHi alkalinization. Additionally, revR4 parasites presented a proton efflux activity similar to that of R4 parasites (22.63 ± 0.37 nmol H+/min per 108 cells). Experiments to determine the recovery capacity of pHi following acidification support these findings. Thus, as can be seen from Table Table3,3, R4 parasites recovered their pHi significantly faster than WT parasites (recovery rates of 0.32 ± 0.04 and 0.17 ± 0.02, respectively). Similar results were obtained for revR4 parasites, which presented a recovery rate intermediate between those for WT and R4 parasites (0.28 ± 0.02; Table Table3).3). The recovery rates for the different Leishmania lines were slightly lower when using the specific H+-ATPase inhibitor DCCD, and a significant decrease in the final pHi was also observed. These results support the presence of a plasma membrane H+-ATPase as a major pHi regulator (15). The pHi recovery was also determined by using chloride-free buffer, as the chloride ion is essential for acid extrusion in Leishmania promastigotes (29). The recovery rates for the R4 and revR4 Leishmania lines were found to be higher than that for WT parasites under these conditions (Table (Table33).

TABLE 3.
pHi recovery in L. major linesa

The effect of TFQ on ATP synthesis.

We assessed the ATP levels in L. major lines in the presence of TFQ and found that WT parasites exhibit a significant decrease in total ATP levels in response to different TFQ concentrations (Fig. (Fig.3A).3A). Furthermore, R4 parasites showed significantly higher basal ATP levels than WT parasites, along with a lower decrease in ATP levels in response to TFQ treatment (Fig. (Fig.3A).3A). Additionally, revR4 parasites presented ATP levels intermediate between those for the WT and R4 lines, although the levels were similar to those for WT parasites at TFQ concentrations above 5 μM (Fig. (Fig.3A).3A). We also examined whether the increased total ATP levels observed for R4 parasites were due to an increase in mitochondrial ATP synthesis or increased glycolysis. Sodium azide treatment inhibits the F1-ATPase and cytochrome c oxidase from complex IV, thus producing a loss of mitochondrial membrane potential that is critical for the electron transport chain activity involved in the generation of ATP. Under our experimental conditions, sodium azide decreased the ATP levels in all Leishmania lines studied, although the ATP levels in R4 parasites were significantly higher than those observed for the WT and revR4 lines (Fig. (Fig.3B),3B), thus suggesting that glycolytically generated ATP is higher in R4 parasites. We therefore inhibited glycolysis in Leishmania lines deprived of glucose but provided with 2-deoxy-d-glucose, a competing substrate for hexokinase, and pyruvate to ensure that the majority of ATP generation was dependent on mitochondrial electron transport and the function of complex V. This inhibition of glycolytic ATP synthesis significantly reduced ATP levels to reach similar values in all Leishmania lines (Fig. (Fig.3B3B).

FIG. 3.
Effect of TFQ on ATP levels in L. major promastigotes. Promastigotes (4 × 106 per ml) of WT (black histograms), R4 (gray histograms), and revR4 (white histograms) L. major lines were incubated (A) in culture medium with different concentrations ...

Increased ATP synthesis by glycolysis in R4 parasites.

The TFQ-resistant Leishmania line upregulates glycolysis, whose final step involves the conversion of phosphoenolpyruvate into pyruvate by the enzyme pyruvate kinase. The change in intracellular pyruvate concentration is therefore proportional to the glycolytic activity. We compared the levels of pyruvate between Leishmania lines using a highly sensitive Amplex Red-based fluorescent assay. As shown in Fig. Fig.4A,4A, the pyruvate concentration in R4 parasites was higher (approximately 3-fold) than that observed for the WT line, in agreement with the higher levels of glycolytically generated ATP. The revR4 parasites presented pyruvate levels intermediate between those for the WT and R4 lines. Furthermore, when parasites were pretreated with the glycolysis inhibitor 2-deoxy-d-glucose, the amount of pyruvate in the three lines dropped significantly (Fig. (Fig.4A),4A), thereby supporting the hypothesis that the higher pyruvate levels present in the R4 line result from glycolysis. As pyruvate kinase activity is known to be critical in glycolysis, we determined this activity in Leishmania lines; no significant differences between parasite lines were observed (data not shown). Furthermore, as trypanosomatids normally use glucose as their main carbon source, we tested whether R4 parasites increased their glucose uptake as the starting product for glycolysis. Thus, the parasites were cultured in the presence of 0.5 mM [14C]glucose for 10 min, and analysis of the uptake of [14C]glucose showed no significant differences between the different Leishmania lines (data not shown). Additionally, to discard the possibility that the observed higher level of ATP produced by glycolysis in the R4 line could be due to a different pHi, which could influence the activity of key enzymes involved in glycolysis, we determined the ATP levels for the Leishmania WT line with different pHi values. The results showed that total ATP values were similar at the different Leishmania pHi values studied (Fig. (Fig.4B4B).

FIG. 4.
Intracellular pyruvate values and the effect of pHi on ATP levels. (A) Intracellular pyruvate levels for WT (black), R4 (gray), and revR4 (white) lines pretreated without (Control) or with (DEO) 2-deoxy-d-glucose were determined by a fluorimetric assay, ...

TFQ induces a decrease in pHi.

As TFQ induces a reduction in intracellular ATP levels and the plasma membrane H+-ATPase is the major regulator of pHi in Leishmania, we investigated whether TFQ treatment modified the pHi of parasites. The results of this study showed that TFQ produces a significant and rapid acidification of pHi after 1 min of treatment (Fig. (Fig.5A).5A). Indeed, after 30 min, the pHi values for WT and revR4 parasites were 6.50 and 6.49, respectively, whereas the acidification induced by TFQ in R4 parasites was significantly lower (pHi 6.94) (Fig. (Fig.5A).5A). These results support our hypothesis that R4 parasites increase their ATP synthesis in order to maintain the functionality of a plasma membrane H+-ATPase involved in the regulation and recovery of pHi as a defensive strategy against the TFQ toxicity. Similar results were observed after treatment with sodium azide (Fig. (Fig.5B),5B), which produces a reduction in pHi as a consequence of ATP depletion and nonoptimal functioning of the plasma membrane H+-ATPase in Leishmania.

FIG. 5.
Acidification of pHi by TFQ and sodium azide in L. major lines. WT (diamonds), R4 (squares), and revR4 (triangles) BCECF-loaded promastigotes were incubated with 5 μM TFQ (A) or with 20 mM sodium azide (B) at different time points (1, 3, 5, 10, ...

DISCUSSION

8-Aminoquinolines, such as sitamaquine and TFQ, have recently been reported to be promising antileishmania drugs (12, 33, 34). Furthermore, we have shown that TFQ induces mitochondrial dysfunction in Leishmania, with the resulting decreased oxygen consumption and depolarization of the mitochondrial membrane potential leading to a final apoptosis-like process (3). In this study, we induced experimental resistance to TFQ in order to determine the ability of Leishmania to generate resistance to this 8-aminoquinoline and as a strategy to validate the mechanism of action of these compounds in this protozoan parasite. Thus, we selected for a TFQ-resistant L. major (R4) line that presents 2.5-fold higher EC50s for TFQ in both the promastigote and intracellular amastigote stages, with an unstable resistant phenotype after 1 month without drug pressure. Experiments to increase the TFQ resistance level further were unsuccessful, although it should be noted that other authors have obtained 5- and 3-fold-higher resistance levels for sitamaquine in the promastigote and intracellular amastigote forms of Leishmania, respectively (2). None of the standard leishmanicidal drugs (SbIII, amphotericin B, miltefosine, and paromomycin) displayed a cross-resistance profile with TFQ in the R4 line; cross-resistance was limited to other 8-aminoquinolines, such as sitamaquine and primaquine. The cross-resistance to sitamaquine detected is interesting, as this is a promising oral drug against leishmaniasis.

Reduced drug uptake is one of the main mechanisms of resistance in Leishmania (2, 5, 19); however, this mechanism was not relevant for the resistance to TFQ, as decreased TFQ uptake levels were similar for the R4 and revR4 lines.

Similarly to sitamaquine (14) and other aminoquinolines (9), TFQ appears to cross the plasma membrane by a pH gradient-driven diffusion process. The R4 parasites present a higher pHi than WT parasites, as does the revR4 line, thus suggesting that these parasites maintain a phenotype with characteristics similar to that of the resistant line even though the TFQ sensitivity was identical to that for WT parasites. The increase of pHi in R4 and revR4 Leishmania lines could contribute to the lower TFQ accumulation observed for these parasites. Indeed, the change in pHi observed as a result of pretreatment with NH4Cl resulted in a significant reduction in TFQ accumulation. Additionally, we observed that R4 and revR4 Leishmania lines have a higher capacity to regulate the pHi than the WT line, although this regulation is not sufficient to prevent the toxicity of this drug in revR4.

Under physiological conditions, approximately 70% of the total bioenergetic requirements of Leishmania are fulfilled by oxidative phosphorylation in the mitochondria, a metabolic process which produces more ATP molecules from a given amount of glucose than glycolysis. However, when the ability of parasites to generate ATP through mitochondrial oxidative phosphorylation is compromised, parasites are able to adapt alternative metabolic pathways, such as increasing their glycolytic activity, to maintain their energy supply. We have demonstrated that an increase in glycolytic metabolism observed for R4 parasites is associated with increased ATP delivery to essential ATP-consuming cell processes, such as the maintenance of ion-motive ATPases required to retain their pHi values. A similar situation has been described for cancer cells, where mitochondrial metabolic defects due, in part, to mutations in mitochondrial DNA, dysfunction of the electron transport chain, aberrant expression of enzymes involved in energy metabolism, and insufficient oxygen available in the cellular microenvironment contribute to an increased dependency on glycolysis (32). This results in increased expression of enzymes required for glycolysis, such as hexokinase II, the enzyme catalyzing the first step of the glycolytic pathway (16). Leishmania R4 parasites can increase glycolytic ATP synthesis in several different ways, including upregulation of glycolytic enzymes or an increased uptake of glucose as a carbon source. Additionally, the modification of pHi in the R4 line can also change the enzymatic activities of glycolytic enzymes. We have observed that Leishmania R4 parasites present an increased accumulation of pyruvate as the end product of glycolysis but with no modification of their pyruvate kinase activity, this kinase being one of the key enzymes involved in glycolysis in trypanosomatids (26). Additionally, R4 parasites do not modify the accumulation of glucose as a carbon source. Thus, Leishmania R4 parasites can increase glycolytic ATP either through upregulation of the metabolism using other substrates as a carbon and energy source or by upregulation of other glycolytic enzymes. The first option can be ruled out in light of the fact that the ATP levels after treatment with sodium azide and deoxyglucose, which inhibits both mitochondrial synthesis and glycolysis, are significantly diminished. This indicates that R4 parasites cannot make use of other carbon sources, such as β-oxidation of fatty acids or the catabolism of certain amino acids (22). Experiments are under way to determine which glycolytic enzymes are upregulated and could therefore be used as molecular markers of TFQ resistance in Leishmania. Additionally, further studies using a metabolomics-based approach will be undertaken to identify the metabolic pathways associated with TFQ resistance.

Acknowledgments

This work was supported by Spanish grants SAF2009-07440 (to F.G.), ISCIII-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET) RD06/0021/0002 (to F.G.), and MSC-FIS PI081902 (to J.M.P.-V.) and by the Plan Andaluz de Investigación (code BIO130).

We acknowledge the support of GlaxoSmithKline (Greenford, United Kingdom) for the tafenoquine, sitamaquine, and [14C]sitamaquine used throughout this research work.

We thank Roberto Docampo for his useful suggestions for the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 January 2011.

REFERENCES

1. Berman, J. D., and L. S. Lee. 1983. Activity of 8-aminoquinolines against Leishmania tropica within human macrophages in vitro. Am. J. Trop. Med. Hyg. 32:753-759. [PubMed]
2. Bories, C., S. Cojean, F. Huteau, and P. M. Loiseau. 2008. Selection and phenotype characterisation of sitamaquine-resistant promastigotes of Leishmania donovani. Biomed. Pharmacother. 62:164-167. [PubMed]
3. Carvalho, L., et al. 2010. Tafenoquine, an antiplasmodial 8-aminoquinoline, targets Leishmania respiratory complex III and induces apoptosis. Antimicrob. Agents Chemother. 54:5344-5351. [PMC free article] [PubMed]
4. Chiquero, M. J., et al. 1998. Altered drug membrane permeability in a multidrug-resistant Leishmania tropica line. Biochem. Pharmacol. 55:131-139. [PubMed]
5. Croft, S. L., S. Sundar, and A. H. Fairlamb. 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19:111-126. [PMC free article] [PubMed]
6. Guerin, P. J., et al. 2002. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect. Dis. 2:494-501. [PubMed]
7. Reference deleted.
8. Hale, B. R., et al. 2003. A randomized, double-blind, placebo-controlled, dose-ranging trial of tafenoquine for weekly prophylaxis against Plasmodium falciparum. Clin. Infect. Dis. 36:541-549. [PubMed]
9. Hawley, S. R., P. G. Bray, B. K. Park, and S. A. Ward. 1996. Amodiaquine accumulation in Plasmodium falciparum as a possible explanation for its superior antimalarial activity over chloroquine. Mol. Biochem. Parasitol. 80:15-25. [PubMed]
10. Jackson, P. R., et al. 1986. Detection and characterization of Leishmania species and strains from mammals and vectors by hybridization and restriction endonuclease digestion of kinetoplast DNA. Vet. Parasitol. 20:195-215. [PubMed]
11. Kennedy, M. L., et al. 2001. Chemosensitization of a multidrug-resistant Leishmania tropica line by new sesquiterpenes from Maytenus magellanica and Maytenus chubutensis. J. Med. Chem. 44:4668-4676. [PubMed]
12. Kinnamon, K. E., et al. 1978. The antileishmanial activity of lepidines. Am. J. Trop. Med. Hyg. 27:751-757. [PubMed]
13. Krogstad, D. J., et al. 1987. Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance. Science 238:1283-1285. [PubMed]
14. López-Martin, C., J. M. Pérez-Victoria, L. Carvalho, S. Castanys, and F. Gamarro. 2008. Sitamaquine sensitivity in Leishmania species is not mediated by drug accumulation in acidocalcisomes. Antimicrob. Agents Chemother. 52:4030-4036. [PMC free article] [PubMed]
15. Marchesini, N., and R. Docampo. 2002. A plasma membrane P-type H(+)-ATPase regulates intracellular pH in Leishmania mexicana amazonensis. Mol. Biochem. Parasitol. 119:225-236. [PubMed]
16. Mathupala, S. P., A. Rempel, and P. L. Pedersen. 2001. Glucose catabolism in cancer cells: identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J. Biol. Chem. 276:43407-43412. [PubMed]
17. Moore, E. M., and D. N. Lockwood. 2010. Treatment of visceral leishmaniasis. J. Glob. Infect. Dis. 2:151-158. [PMC free article] [PubMed]
18. Mullin, K. A., et al. 2001. Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana. Mol. Biol. Cell 12:2364-2377. [PMC free article] [PubMed]
19. Ouellette, M., J. Drummelsmith, and B. Papadopoulou. 2004. Leishmaniasis: drugs in the clinic, resistance and new developments. Drug Resist. Updat. 7:257-266. [PubMed]
20. Reference deleted.
21. Sandoval, W., R. Isea, E. Rodriguez, and J. L. Ramirez. 2008. A biochemical and genetic study of Leishmania donovani pyruvate kinase. Gene 424:25-32. [PubMed]
22. Saunders, E. C., et al. 2010. Central carbon metabolism of Leishmania parasites. Parasitology 137:1303-1313. [PubMed]
23. Seifert, K., et al. 2003. Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int. J. Antimicrob. Agents 22:380-387. [PubMed]
24. Shanks, G. D., et al. 2001. A new primaquine analogue, tafenoquine (WR 238605), for prophylaxis against Plasmodium falciparum malaria. Clin. Infect. Dis. 33:1968-1974. [PubMed]
25. Tekwani, B. L., and L. A. Walker. 2006. 8-Aminoquinolines: future role as antiprotozoal drugs. Curr. Opin. Infect. Dis. 19:623-631. [PubMed]
26. van Schaftingen, E., F. R. Opperdoes, and H. G. Hers. 1985. Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate. Eur. J. Biochem. 153:403-406. [PubMed]
27. Verdier, F., J. Le Bras, F. Clavier, I. Hatin, and M. C. Blayo. 1985. Chloroquine uptake by Plasmodium falciparum-infected human erythrocytes during in vitro culture and its relationship to chloroquine resistance. Antimicrob. Agents Chemother. 27:561-564. [PMC free article] [PubMed]
28. Vieira, L., A. Lavan, F. Dagger, and Z. I. Cabantchik. 1994. The role of anions in pH regulation of Leishmania major promastigotes. J. Biol. Chem. 269:16254-16259. [PubMed]
29. Vieira, L., I. Slotki, and Z. I. Cabantchik. 1995. Chloride conductive pathways which support electrogenic H+ pumping by Leishmania major promastigotes. J. Biol. Chem. 270:5299-5304. [PubMed]
30. Walsh, D. S., et al. 2004. Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P. falciparum malaria. J. Infect. Dis. 190:1456-1463. [PubMed]
31. Walsh, D. S., et al. 2004. Randomized trial of 3-dose regimens of tafenoquine (WR238605) versus low-dose primaquine for preventing Plasmodium vivax malaria relapse. Clin. Infect. Dis. 39:1095-1103. [PubMed]
32. Xu, R. H., et al. 2005. Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res. 65:613-621. [PubMed]
33. Yardley, V., F. Gamarro, and S. L. Croft. 2010. Antileishmanial and antitrypanosomal activity of the 8-aminoquinoline tafenoquine. Antimicrob. Agents Chemother. 54:5356-5358. [PMC free article] [PubMed]
34. Yeates, C. 2002. Sitamaquine (GlaxoSmithKline/Walter Reed Army Institute). Curr. Opin. Investig. Drugs 3:1446-1452. [PubMed]
35. Zhu, A., R. Romero, and H. R. Petty. 2010. A sensitive fluorimetric assay for pyruvate. Anal. Biochem. 396:146-151. [PMC free article] [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...