Probenecid, prochlorperazine, verapamil, potassium antimonyl tartrate hydrate, kanamycin, L-buthionine-(SR)-sulfoximine (BSO), sodium azide (NaN₃), HEPES, biopterin, ampicillin, L-glutamine and hemin were obtained from Sigma-Aldrich (St Louis, USA). Nitric acid (65%) was obtained from Merck Brasil (Rio de Janeiro, RJ, Brazil).

Promastigote forms of two different New World Leishmania species: Leishmania (Viannia) guyanensis (MHOM/BR/1975/M4147) and Leishmania (Viannia) braziliensis (MHOM/BR/1975/M2904) were used. Parasites of both strains were selected in vitro for resistance to Sb^III as previously described (Roberts and Rainey, 1993; Moreira et al., 2013). Promastigotes were exposed to increasing Sb^III concentrations up to 650 μM (L. guyanensis) and 330 μM (L. braziliensis) in 25 cm² flasks containing 5 mL of minimum essential culture medium (α-MEM) (Gibco, Invitrogen, NY, USA). The selected parasite strains (L. braziliensis Sb^III330.2 and L. guyanensis Sb^III650.4) were maintained in α-MEM, supplemented with 10% (v/v) heat-inactivated fetal calf serum (Cultilab, Brasil), 100 μg/mL kanamycin, 50 μg/mL ampicillin, 2 mM L-glutamine, 5 μg/mL hemin, 5 μM biopterin, pH 7.0 and incubated at 25°C in a B.O.D. incubator. As previously described, the resistant mutant L. guyanensis Sb^III650.4 harbors a single nucleotide polymorphism at AQP1 coding gene that leads to the point mutation G133D at protein level. Functional analysis revealed that this mutation was directly associated with the reduced antimony uptake (Monte-Neto et al., 2015). On the other hand, L. braziliensis resistant mutant presented intact copies of AQP1 as revealed by gene sequencing (Supplementary Figure 1).

To compare the Sb^III sensitivity of different strains, mid-log phase wild-type and resistant Leishmania promastigotes were inoculated at 10⁶ cells/mL in α-MEM medium in the presence of Sb^III (as potassium antimonyl tartrate). Biological replicates in the absence of drug were established as control. The cultures were incubated under shaking at 25 ± 1°C for 72 h and the growth inhibition was determined by measuring the absorbance at 600 nm using a microplate reader (Organon Teknica Microwell), as previously described (Fumarola et al., 2004). Three independent experiments were carried out. The half-maximal inhibitory concentration (IC₅₀) values were calculated based on concentration-response curves applying a sigmoidal dose-response equation with variable slope carried out using the software GraphPad Prism version 6.0 (GraphPadSoftware Inc., San Diego, CA, USA).

Total RNA was extracted from 10⁸ mid-log phase Leishmania spp. promastigotes using RNeasy Plus mini kit (Qiagen Sciences, Maryland, USA) as described by the manufacturer. First-strand cDNA was synthesized from 2.5 μg of total RNA using Oligo dT12–18 and SuperScript II RNase H-Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer protocol. Equal amounts of cDNA were run in triplicate and amplified in 25 μL reactions containing 1 x iQ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA), 100 nM forward and reverse primers and 100 ng of cDNA target. Reactions were carried out using a rotator thermocycler Rotor Gene (RG 3000, Corbett Research, San Francisco, USA). Mixtures were initially incubated at 95°C for 5 min and then cycled 30 times at 95°, 60°, and 72°C for 15 s. No-template controls were used as recommended. Three technical and biological replicates were established for each reaction. The relative amount of PCR products generated from each primer set was determined based on the cycle threshold (Ct) value and the amplification efficiencies. Data were analyzed using the comparative 2^−ΔΔCt method. Gene expression levels were normalized to constitutively expressed mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH, LbrM.30.2950). The primers for targeted genes: MRPA (LbrM.23.0280), GSH1—that encodes to gamma-glutamylcysteine synthetase (γGCS-LbrM.14.0880), ODC (LbrM.12.0300), AQP1 (LbrM.31.0020), ABCI4 (LbrM.33.3540), ARM58 (LbrM.20.0210), and internal gene expression control GAPDH were designed using PrimerQuest® (https://www.idtdna.com/Primerquest/Home/Index). Primer sequences are listed in Table 1.

Total intracellular thiols were derivatized from deproteinized cell extracts and separated by high-performance liquid chromatography (HPLC) as previously described (Fairlamb et al., 1987; Mukhopadhyay et al., 1996). Briefly, 10 mL of Sb-free logarithmic phase Leishmania promastigote cultures were haversted at 0.3–0.4 absorbance (600 nm); washed twice in HEPES/NaCl (21 mM HEPES; 137 mM NaCl; 5 mM KCl; 0.7 mM Na₂ HPO₄; 6 mM Glucose; pH 7) and resuspended in HEPES/EDTA (50 mM HEPES; 5 mM EDTA; 1 mM DTT pH 8). At this step, 10 μL were separated for protein dosage by bradford method (Bradford, 1976). Protected from light, 100 μL of 2 mM monobromobimane (mBBr) (Invitrogen, Carlsbad, CA, USA) were added to the samples, mixed and incubated at 70°C for 3 min. Trichloroacetic acid (Fischer Chemical, Atlanta, GA, USA) (200 μL at 25%) were added to the mixture, and the extract was kept at 80°C for at least, 1 h followed by low temperature (4°C) centrifugation at top speed (microcentrifuge) for 20 min. Supernatants were filtered in 0.45-μm filters (Acrodisc Pall, Life Sciences, East Hills, NY, USA) and thiols separated using the liquid chromatograph Shimadzu SCL 10A. Samples were analyzed using a reverse phase column Vydac C18 eluted in methanol 0–100%/acetic acid (25%) gradient pH 3.5. Standard solutions of mBBr-derivatized cysteine (Cys), glutathione (GSH) and trypanothione (TSH) were previously established as calibration curve. Thiols were indirectly measured by mBBR fluorescence at 360 and 450 nm of excitation and emission, respectively, using a coupled fluorescence detector (Shimadzu RF-10Axl).

Before performing the assays, Sb^III-resistant Leishmania spp. lines were maintained for at least two passages in α-MEM medium in the absence of Sb^III, in order to eliminate the residual drug.

The Sb uptake kinetic and influx assays were based on previously described protocols (Roberts and Rainey, 1993; Moreira et al., 2013). Briefly, mid-log phase wild-type and resistant Leishmania promastigotes were washed twice in Hepes/Glucose (HG) buffer (20 mM HEPES, 0.15 M NaCl, 10 mM glucose, pH 7.2) and suspended in this buffer at a density of 10⁸ cells/mL.

In the uptake kinetic assay, cells were incubated in the presence of Sb^III at 540 μM. In different time points, 1 mL of the cell suspension was harvested and immediately centrifuged at 3,000 × g for 5 min at 4°C and the pellet was washed twice with HG buffer under the same conditions. The pellet was then resuspended in 100 μL HG buffer. A 10-μL aliquot of each sample was used for parasite quantification and viability evaluation and the remaining volume (90 μL) was submitted to digestion in nitric acid (65%). Cell viability was confirmed from the promastigote motility and trypan blue exclusion assay (Freshney, 1994). More than 90% of promastigotes showed motility, except those exposed to sodium azide and more than 95% of cells were considered viable according to trypan blue exclusion. The Sb concentration was determined by graphite furnace atomic absorption spectroscopy (Perkin Elmer, AAnalyst 600). The signal from a blank (cells without Sb) was used for background subtraction. The analytical method for determination of Sb was validated and showed suitable levels of precision, accuracy and linearity. The quantification limit of the analytical method was 0.021 nmol Sb/108 promastigotes. The amount of cellular Sb at the zero time point was equal to 0.044 ± 0.002 nmol Sb/108 promastigotes and, thus, close to the quantification limit, indicating that the binding of Sb to the cell surface is negligible, in agreement with the high hydrophilicity of potassium antimonyl tartrate.

For influx assay, parasites were exposed to Sb^III for 1 h at 25°C, at increasing concentrations (0; 100; 250; 500; 1,000; 1,500; 2,000 μM of Sb^III), in quadruplicates/point. Samples were submitted to the washing procedure mentioned above for removing extracellular Sb traces. Each influx assay was performed three times. The influx rate was calculated as follows: Vi = amount of intracellular antimony/(number of cells x time of uptake). A shorter incubation time could not be used, because of the method quantification limit and loss of accuracy in the determination of the initial rate. Thus, the values determined for initial rate were approximation of the influx rates, especially in the case of WT cells in which Sb uptake was not linear over 1-h time. The Vi-vs-Ce curves were analyzed using the GraphPad Prism 6.0 software to assess whether they best fit with a linear model or the Michaelis-Menten equation as follows:

where V_max is the maximum influx rate, K_m is the Michaelis-Menten constant and Ce is the extracellular concentration of Sb.

The influx rate constant (k_influx) was calculated as:

When no saturation was observed, k_influx was determined by linear regression, assuming that Vi = k_influx × Ce.

This influx assay was also used to identify the values of Ce at which the mutant and its parental line exhibited the same intracellular concentration of Sb, to be explored in the efflux protocol.

To evaluate the effect of ABC transporter inhibitors on Sb^III uptake, mid-log phase Leishmania spp. promastigotes were first exposed for 24 h to each of these compounds at non-toxic concentrations (8 μM verapamil, 4 mM probenecid, 10 μM prochlorperazine, 100 μM BSO for resistant strains and 8 μM verapamil, 4 mM probenecid, 3.5 μM prochlorperazine, 100 μM BSO for parental strains). The cells were then resuspended in HG buffer at 10⁸ cells/mL and further exposed to these compounds for 1 h at 25°C, in the presence of 1 mM of Sb^III. Samples were washed twice with cold HG buffer to remove external Sb and allow the measurement of intracellular Sb. We observed that more than 90% of promastigotes showed motility at the end of the experiment, suggesting that the inhibitors did not act through depletion of intracellular ATP.

When investigating the impact of energy depletion on the uptake of Sb^III, cells were resuspended in the HG buffer without glucose, but in the presence of 10 mM sodium azide. Those were then incubated for 1 h at 25°C in the presence of 1 mM of Sb^III and were subsequently processed as described above.

Before performing this assay, Sb^III-resistant Leishmania lines were maintained for at least two passages in α-MEM medium in the absence of Sb^III, in order to remove the residual drug. Mid-log phase wild-type and resistant Leishmania promastigotes were washed twice with HG buffer and resuspended in it at a density of 10⁸ cells/mL. A 1-mL aliquot containing only parasites (blank) was separated and the remaining cells were incubated at 25°C with the concentration of Sb^III pre-established in influx assay so as to obtain the same loading of drug in the resistant strain and its parental cells (L. guyanensis, 100 μM for WT and 2,000 μM for the mutant; L. braziliensis, 500 μM for WT and 2,000 μM for the mutant). After 1 h incubation, the cells were centrifuged at 3,000 × g for 5 min at 4°C, washed twice and resuspended in HG buffer at the original cell density followed by incubation at 25°C. Aliquots of 1 mL were taken from the parasite suspension at 0, 15, 30, 60, and 120 min. Subsequently, these aliquots and the blanks were treated as described above for the quantification of parasite and intracellular Sb. The signal from blanks was considered as background.

Each efflux assay was performed three times in triplicate. Data were plotted as the percentage of initial intracellular Sb content as a function of time and the half-time of Sb efflux was calculated using mono-exponential decay model. The efflux rate constant k_efflux was also obtained from the equation:

where Ve is the initial rate of efflux determined from the tangent of the curve and Ci is the intracellular concentration of Sb estimated at time zero using a cell volume of 1.2 × 10⁻¹⁴ L (Zilberstein and Dwyer, 1984).

To evaluate the interference of ABC transporter inhibitors on Sb efflux, the cells were initially loaded for 1 h at 25°C with 1 mM of Sb^III in α-MEM medium. The cells were washed and resuspended in HG buffer at 10⁸ cells/mL. A 1-mL aliquot was immediately removed and processed to determine the initial intracellular amount of Sb^III. The remaining cells were exposed to the inhibitors at non-toxic concentrations (8 μM verapamil, 4 mM probenecid, 10 μM prochlorperazine, 100 μM BSO, 10 mM sodium azide in resistant strains and 8 μM verapamil, 4 mM probenecid, 3.5 μM prochlorperazine, 100 μM BSO, 10 mM sodium azide in parental strains) for 2 h at 25°C under agitation. Only when evaluating the effect of azide, that the buffer did not contain glucose. The cells were subsequently processed as described above to determine the amount of Sb per cell. We observed that more than 90% of promastigotes showed motility at the end of the experiments when applying verapamil, probenecid, prochlorperazine or BSO, suggesting that these inhibitors did not act through depletion of intracellular ATP.

First, ABC transporter inhibitors (probenecid, prochlorperazine, verapamil and BSO) were evaluated for their antileishmanial activity against SbR and WT Leishmania spp. Mid-log phase promastigotes were inoculated at 10⁶ cells/mL in α-MEM medium in the presence of different concentrations of the inhibitors. Biological replicates in the absence of drug were established as control. The cultures were incubated under shaking at 25 ± 1°C for 72 h and the IC₅₀s were determined as described above (section Leishmania Strains and Sb^III Sensitivity Assay). The ability of each inhibitor to sensitize the cells to Sb^III was evaluated by performing growth inhibition assay as described above, in the presence of a fixed non-toxic concentration of the inhibitor (8 μM verapamil, 4 mM probenecid, 10 μM prochlorperazine, 100 μM BSO in resistant strains and 8 μM verapamil, 4 mM probenecid, 3.5 μM prochlorperazine, 100 μM BSO in parental strains). The IC₅₀ values of Sb^III in presence and absence of inhibitor were compared. All experiments were done at least three times as independent experiments performed in triplicate.

The IC₅₀ values were calculated by non-linear regression. Data were analyzed by Student's t-test or One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. A p ≤ 0.05 was considered statistically significant. All analyses were carried out using the software GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA).

While L. braziliensis and L. guyanensis parental strains presented IC₅₀ of Sb^III lower than 100 μM, the resistant mutants suffered little influence of Sb^III at concentration as high as 600 μM (Table 2 See also growth inhibition curves in Supplementary Figure 2).

The mRNA levels of MRPA, GSH1, ODC, AQP1, ABCI4, and ARM58—genes associated with Sb resistance in Leishmania parasites—were investigated. As shown in Figure 1B, increased mRNA levels of MRPA (2-fold) and to a lower extent of GSH1 (1.5-fold) were found in LgSbR mutant, when compared to the parental strain. No differences were observed for ODC or AQP1 mRNA for this mutant (Figure 1). On the other hand, LbSbR mutant did not exhibit any relevant change in mRNA levels of either MRPA, GSH1, AQP1, ABCI4, or ARM58 (Figure 1A). Despite the fact that no significant difference on mRNA levels of thiol biosynthetic enzymes was observed, increased levels of cysteine and glutathione for LgSbR and a higher amount of trypanothione in LbSbR were detected when compared to WT lines (Figure 2).

Figure 3 shows the kinetics of Sb uptake in L. braziliensis and L. guyanensis SbR mutants in comparison to their parental strains. The results indicate that both mutants exhibited a lower initial rate of Sb influx (3 × 10⁻¹² and 0.4 × 10⁻¹² nmol.s⁻¹.cell⁻¹ for L. braziliensis and L. guyanensis, respectively) when compared to their respective susceptible counterparts (7 × 10⁻¹² and 5 × 10⁻¹² nmol.s⁻¹.cell⁻¹, for L. braziliensis and L. guyanensis, respectively). As illustrated in Figure 4, the determination of the initial rate of Sb influx as a function of the extracellular Sb concentration showed a saturation at high drug concentration in the case of all strains, except for LgSbR. The kinetics constants of Sb influx were then calculated according to the Michaelis-Menten model (Table 3). We can infer that the decrease of drug influx contributed to the reduction of drug uptake, mainly in the case of LgSbR.

After loading the mutant and WT promastigotes with a similar amount of Sb (the initial amounts of Sb were respectively: 0.50 ± 0.018 nmol/10⁸ cells and 0.53 ± 0.021 nmol/10⁸ cells for L. guyanensis WT and mutant; and 2.37 ± 0.53 nmol/10⁸ cells and 2.28 ± 0.61 nmol/10⁸ cells for L. braziliensis WT and mutant), the cells were washed and resuspended in a drug-free buffer to assess the kinetic of drug release. As shown in Figure 5, the Sb efflux was faster from the SbR mutants when compared with their WT counterparts. Assuming a monoexponential drug release model, the half-time of drug release was estimated and compared between the different cell lines (Table 4). The efflux of Sb was found 45-fold and 21-fold faster in LgSbR and LbSbR, respectively. These data support the model in which the increase of drug efflux strongly contributes to the reduction of drug uptake in both Leishmania mutants.

To further characterize the Sb^III transport routes involved in the resistance of both L. braziliensis and L. guyanensis Sb- resistant mutants, a pharmacological approach was adopted based on the use of the following classical ABC transporter inhibitors: verapamil as MDR1-transporter inhibitor (Neal et al., 1989; Valiathan et al., 2006); probenicid as MRP-transporter inhibitor (Courtois et al., 1999; Payen et al., 2000; Mandal et al., 2009); prochlorperazine as MDR1- and MRP-transporter modulator (Essodaigui et al., 1999; Wesołowska, 2011; Rai et al., 2013) and BSO as intracellular thiol-depleting agent (Arana et al., 1998).

Table 5 displays the sensitivity (IC₅₀) of the L. braziliensis and L. guyanensis strains to the different ABC transporter inhibitors (See also growth inhibition curves in Supplementary Figure 3). Although most of the inhibitors exhibited IC₅₀ values in the same range when comparing the WT and SbR strains, prochlorperazine showed a distinct profile, as it was about 20-fold more active against the WT than LgSbR. This apparent cross-resistance suggests that prochlorperazine and Sb^III may share the same transport pathway in this mutant. Verapamil was also 2-fold more active against the WT than LgSbR, however, it showed an opposite profile in the L. braziliensis strains.

The antileishmanial activities of Sb^III in the absence and presence of ABC transporter inhibitors were compared, for each tested Leishmania strain (Table 6 See also growth inhibition curves in Supplementary Figures 4,5). Among the different inhibitors, prochlorperazine was the only agent to resensitize both SbR strains, the most pronounced effect being observed in L. guyanensis. Interestingly, no such sensitization was observed in the WT parental strains. However, the lower concentration of prochlorperazine used in the WT lines (because of their greater susceptibility) may explain the lack of sensitization. Probenecid specifically sensitized the L. braziliensis strains to Sb^III, but this effect occurred in both the mutant and wild-type strains. Surprisingly, verapamil promoted sensitization to Sb^III specifically in the WT strains.

Figure 6 shows the impact of cell pre-exposure to ABC transporter inhibitors on the subsequent Sb uptake. Prochlorperazine was the only inhibitor to enhance Sb uptake specifically in SbR strains, in agreement with its sensitizing effect. On the other hand, probenecid enhanced the uptake of Sb only in LgSbR. It is noteworthy that BSO resulted in increased Sb uptake in LgSbR mutant and in both WT and LbSbR. In contrast, exposition to the metabolic inhibitor sodium azide did not promote significant change in Sb uptake in any of the tested cell lines. Also, corroborating the results of the sensitization assay, verapamil increased the Sb uptake in the WT strains of both species.

To evaluate the effect of the inhibitors on the efflux of Sb, cells were first exposed for 1 h to 1 mM Sb^III, washed, resuspended in drug-free Hepes/NaCl buffer and incubated for 2 h in the absence or presence of the inhibitor. Figure 7 displays the percentages of Sb released from the different strains after 2 h of efflux. Prochlorperazine was the only agent to significantly inhibit Sb efflux specifically in the resistant strains, in agreement with its ability to increase the cellular drug uptake. Probenecid reduced the efflux of Sb in both mutants, but the effect was only significant in LbSbR. Sodium azide markedly inhibited the efflux of Sb mainly in SbR mutants, evidencing that Sb efflux in the resistant strains is essentially energy-dependent. The thiol-depleting agent BSO showed a significant effect only in LgSbR. On the other hand, verapamil exerted no significant effect on the drug efflux.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.