Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

Clinical isolates.

Clinical isolates of VL were obtained from the splenic aspirates of AmB-responsive and -nonresponsive patients in the indoor ward facility of Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India. Briefly, the collected splenic aspirates were incubated in RPMI-1640 medium (Gibco) (pH 7.4) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% of penicillin (50 U/ml)-streptomycin (50 mg/ml) solution (Sigma) at 25°C. The amastigotes from splenic aspirates were transformed into promastigotes, and they were maintained further in RPMI-1640 medium supplemented with FBS.

Cell line.

The THP1 cell line was maintained in a 25-cm² flask (Corning) in Dulbecco's modified Eagle medium (DMEM) (Sigma) supplemented with 10% FBS and antibiotics (streptomycin and penicillin) in a humidified 5% CO₂/air atmosphere at 37°C.

Characterization and clonal selection of clinical isolates.

Clinical isolates were confirmed as Leishmania spp. by amplification of kinetoplast DNA (kDNA) using a kDNA gene-specific primer (F, 5′-TCTGTGGCCCATTTGTTGTA-3′, and R, 5′-CATTTTTGGGTTTTCGGAGA-3′). The isolates were then clonally selected by growing them on NNN agar slant medium. The single colonies formed on the agar slant were further grown separately in RPMI-1640 medium.

In vitro drug sensitivity assay.

In vitro drug sensitivity was determined by incubating 2 × 10⁶ parasites in RPMI-1640 medium (supplemented with 10% FBS) with different concentrations of AmB and at 1-day intervals for 6 consecutive days. Parasites were not treated with AmB in the control experimental set. Viable cells were counted in a hemocytometer (Rohem) by the trypan blue (Sigma) (0.5 mg ml⁻¹) exclusion method, and the 50% lethal doses (LD₅₀) were determined for both the AmB-resistant and AmB-sensitive strains. There were three replicates in each test, and the data are the means and the standard deviations (SDs) of three experiments.

MTT assay.

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay is a quantitative colorimetric assay for measurement of metabolically active cells. This assay is based on cleavage of the yellow tetrazolium salt, MTT (Sigma), which forms water-insoluble, dark blue formazan crystals, and this cleavage happens in living cells only because of the mitochondrial enzyme succinate dehydrogenase. To determine the LD₅₀ of AmB using an in vitro drug sensitivity assay, 10 μl of MTT solution (5 mg/ml) was added for each 100 μl of untreated or drug-treated parasite culture. After addition of MTT, the cultures were incubated at 22.4°C for 3 h and subsequently incubated with 200 μl of MTT solubilization buffer. Absorbance was recorded at 570 nm using a UV-visible spectrophotometer (Hitachi, Japan). The MTT assay was also performed to quantify the proportion of metabolically active cells after the addition of inhibitors (both the thiol metabolic pathway and ABC transporter inhibitors) and a reactive oxygen species (ROS) scavenger, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (20 mM) (Sigma), for untreated and AmB-treated parasites. There were three replicates in each test, and the data are the means and SDs of three experiments.

Cell cytotoxicity assay.

THP1 cells were counted in an improved Neubauer chamber using the vital stain trypan blue, and 10⁴ cells/well were placed in a 96-well plate with different concentrations of AmB. After 48 h of incubation, the medium was removed, 200 μl of fresh supplemented medium and 20 μl of alamarBlue (Sigma) were added, and the absorbance was measured at 550 nm. There were three replicates in each test, and the data are the means and SDs of three experiments.

Ex vivo assay.

An ex vivo assay was performed using a modification of the method described by Mendez et al. (40). Briefly, 10⁴ THP1 cells/well were cultured in 8-well Lab-Tek chambers (Nunc, Roskilde, Denmark). Once macrophages were adhered, 10⁵ stationary-phase Leishmania promastigotes were added to each well and maintained at 33°C in 5% CO₂ overnight. Noninternalized promastigotes were eliminated, and different concentrations of AmB were added to the wells and incubated for 48 h. Slides were then fixed and stained (Giemsa), and the number of amastigotes/100 cells was determined. LD₅₀ values for both resistant and sensitive parasites were calculated. There were three replicates in each test, and the data are the means and SDs of three experiments.

K^+_i level.

The intracellular K⁺ (K^+_i) levels of AmB-treated (0.125 μg/ml) resistant and sensitive parasites were determined using 5 mM potassium-binding benzofuran isophthalate acetoxymethyl (PBFI-AM; Sigma) as a cell-permeant probe and an LS55 spectrofluorimeter (Perkin Elmer). Fluorescence in the parasites was analyzed by excitation at 370 nm, and emission was registered at 540 nm.

Membrane fluidity.

Diphenylhexatriene (DPH) has been used as a fluorescence probe to measure the fluidity of membranes (34, 35). In brief, a stock solution of the probes (2 mM DPH in tetrahydrofuran) was stored at 4°C in the dark. For labeling, the 2 mM final concentration of DPH was added to both AmB-resistant and AmB-sensitive promastigotes (2 × 10⁶ cells/ml) and incubated for 45 min at 37°C. The suspension was centrifuged, washed, resuspended in phosphate-buffered saline (PBS; pH 7.2), and fixed with 4% paraformaldehyde. The fluorescence anisotropy was immediately measured in an LS55 spectrofluorimeter (Perkin Elmer). The excitation and emission wavelengths were 360 nm and 430 nm, respectively. Fluorescence anisotropy was calculated by the following equation: I_vv − (GF × I_vh)/I_vh + (2 × GF × I_vh). Here, I_vv and I_vh are the intensities with polarizers that are vertical and vertical (excitation and emission, respectively) and vertical and horizontal, respectively. The grating factor (GF) is specific for the instrument and is defined by the ratio of I_hv to I_hh, where I_hv and I_hh are the intensities with polarizers that are horizontal and vertical (excitation and emission, respectively) and horizontal and horizontal, respectively. The polarization value of DPH fluorescence was used as a measure of membrane fluidity, since decreased anisotropy indicates an increase in membrane fluidity (29).

Extraction and analysis of free sterols.

Promastigotes were grown as described above in three Erlenmeyer flasks in a total volume of 1 liter of culture medium. For cell treatment, a volume of the drug corresponding to an appropriate final concentration was added to 10% dimethyl sulfoxide in water, and cultures were incubated at 27°C for various times at 150 rpm in an orbital incubator (Gallenkamp). Cells were harvested, washed, and pooled, and the pellet was resuspended in 20 ml of dichloromethane-methanol (2:1, vol/vol) for about 24 h at 4°C. After centrifugation (11,000 × g, 1 h, 4°C), the extract was evaporated under vacuum. The residue and the pellet were saponified with 30% KOH in methanol at 80°C for 2 h. Sterols were extracted with n-hexane, which was thereafter evaporated, and the residue was dissolved in dichloromethane. An aliquot of clear yellow sterol solution was added to 2 volumes of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and the sealed tubes were heated at 80°C for 1 h. The trimethylsilyl (TMS) ethers of the sterols were subjected to gas chromatography/mass spectrometry (GC/MS) analysis by following previously described methods with some modifications (21, 30). GC was performed with a Varian model 3400 chromatograph equipped with DB5 columns (methyl-phenylsiloxan ratio, 95/5; dimensions, 30 m by 0.25 mm). The gas carrier was He (1 ml/min). Analysis conditions were as follows: the column was kept at 270°C, the injector was kept at 300°C (splitless), and the detector was kept at 300°C (isothermic conditions for sterols). The linear gradient for methyl esters was from 150 to 180°C at 10°C/min. The MS conditions were 280°C, 70 eV, and 2.2 kV.

Determination of intracellular AmB content in treated promastigotes.

AmB-resistant and -sensitive promastigotes were cultured in 25-cm² flasks at an initial concentration of 1 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% FBS. Flasks were kept at 22.5°C with and without AmB (0.125 μg/ml) for the desired time periods. Promastigotes were harvested by centrifugation and washed twice with large volumes of cold PBS (pH 7.2). Pellets were resuspended in an aqueous solution of 20 mM cholic acid for 24 h at 4°C and then centrifuged (11,000 × g, 1 h, 4°C). An equal volume of cold ethanol was added to the supernatant, and the mixture was kept in an ice bath for 1 h to precipitate the proteins. The samples were then centrifuged (11,000 × g, 1 h, 4°C), and the clear supernatants were analyzed by high-performance liquid chromatography (HPLC) (Hitachi) for AmB determination.

HPLC analysis.

Reverse-phase chromatography analysis on a C₁₈ reversed-phase column (4.6 by 250 mm) (Zorbax) was performed with an HPLC (Hitachi). The detection was at 408 nm, corresponding to the maximum absorbance for AmB. An isocratic mobile phase of acetonitrile-water-acetic acid (45:51:5) was delivered at a flow rate of 1 ml/min. Standard concentrations of 0.0125, 0.125, 0.25, 0.5, 1, and 5 μg/μl were made by dissolving AmB in the mobile phase or in methanol.

Drug efflux measurement.

Leishmania promastigotes treated with the drug were harvested as described above and transferred to fresh, drug-free medium after different time points and incubated for 2 h prior to extraction. The AmB efflux was measured using HPLC (Hitachi) in a manner similar to that described above.

Semiquantitative RT-PCR and real-time PCR.

Reverse transcription was performed using 0.2 mg total RNA from untreated L. donovani cells and 0.125 μg/ml AmB-treated L. donovani cells incubated for 8 h using an anchored oligo(dT) (H-dT11M, where M represents A, C, or G; GenHunter). The synthesized cDNAs were amplified by PCR for specific genes, viz., MDR1, PgPA, ODC, spermidine synthase (SPS), γ-GCS, trypanothione synthetase (TryS), TR, cytoplasmic tryparedoxin (cTXN), cytoplasmic tryparedoxin peroxidase (CTP), mitochondrial tryparedoxin (mTXN), mitochondrial tryparedoxin peroxidase (MTP), and S-adenosyl-l-methionine:C-24-Δ-sterolmethyltransferase (SCMT) A and B. The forward (F) and reverse (R) primers of SCMT A and SCMT B were designed on the basis of the 3′ untranslated region (UTR) (48). In all PCRs after initial denaturation at 94°C for 5 min, targeted genes were amplified by 25 amplification cycles (94°C for 30 s, 56°C or 58°C for 30 s, and 72°C for 1 min), followed by a final extension at 72°C for 5 min. All PCRs were performed for 25 cycles, which was within the linear range of amplification of the corresponding mRNA species. The products were run on 1.5% agarose gel, stained with ethidium bromide, and finally documented and quantified using the Bio-Rad gel documentation system and associated Quantity One software. All the reverse transcription PCR (RT-PCR) products were normalized with respect to the alpha-tubulin (a-tub) RT-PCR product. These semiquantitative data were validated by quantitative real-time PCR, which was performed in the LightCycler 480 (Roche) using SYBR green (Roche) chemistry. The cycling conditions were as follows: 1 cycle at 95°C for 3 min and 40 cycles of 95°C for 15 s (denaturation), 58°C for 30 s (annealing), and 72°C for 30 s (extension). The fluorescence signal was captured at the end of each cycle using the SYBR channel (490-nm wavelength for excitation and 525-nm wavelength for emission). Results are the target/reference ratios of each sample, normalized by the target/reference ratio of the calibrator. Here, the target/reference value of sensitive parasites was used as the calibrator and the alpha-tubulin was used as the reference.

The primers used for both semiquantitative RT-PCR and quantitative real-time PCR were as follows: for MDR1, 5′-AATGCTCTTGAGCCTCA-3′ (F) and 5′-CTTCCAGTTGACTACATTCC-3′ (R); for PgPA, 5′-TCAATCAGTCAAATGTCTTGC-3′ (F) and 5′-GAATGTCAACTTTCCTACTCC-3′ (R); for ODC, 5′-GCACGCGCTTCTCATGAACGTATT-3′ (F) and 5′-CGAAGAGGATGCAGTTGAAGCTGT-3′ (R); for SPS, 5′-ACTTCTACACGAATGTGCTCCGCA-3′ (F) and 5′-CATTGCGTACTTGACCGTGGCAAA-3′ (R); for γ-GCS, 5′-AGCGATAAACCGCTCGTACTGTGA-3′ (F) and 5′-ATGTTGTCAAAGTGCTCCGTGTGC-3′ (R); for TryS, 5′-TGTCATGAGCGAATGACCAACCGAT-3′ (F) and 5′-GCTTGCCATTCAACAAACGTCAGGT-3′ (R); for TR, 5′-AATGAGGACGGCTCGAATCACGTT-3′ (F) and 5′-ATGGCGTAGATGTTGTCCACCGAT-3′ (R); for cTXN, 5′-AAGCTAAACACGCAGGTTGTTGCG-3′ (F) and 5′-ATACCGGATTCCTCGATCAGCACA-3′ (R); for CTP, 5′-CCAACGGCAGCTTCAAGAAGATCA-3′ (F) and 5′-TGAAGTCGAGCGGGTAGAAGAAGA-3′ (R); for mTXN, 5′-GCTTCACTCCGAAGCTCGTTGAAT-3′ (F) and 5′-AGTACTCCATGAAAGCGTCGGCCT-3′ (R); for MTP, 5′-AAGCTAAACACGCAGGTTGTTGCG-3′ (F) and 5′-TCGATCAGCACACCATAGTCACGA-3′ (R); for SCMT A, 5′-CATCTTCCCTCCCTTTCCTC-3′ (F) and 5′-CCGCATGAACAACAGAGAGA-3′ (R); and for SCMT B, 5′-TGTCCAGTAGTCCCGGAAAC-3′ (F) and 5′-CGGCGATGATAGTGATGTTG-3′ (R).

Determination of total intracellular reduced thiol content.

The level of total intracellular thiol was measured in deproteinized cell extracts for both resistant and sensitive strains. The late-log-phase cells were harvested, washed with a buffer (0.14 M Na₃PO₄, 0.14 M K₃PO₄, 0.14 M NaCl, and 3 mM KCl) (pH 7.4), and suspended in 0.6 ml of 25% trichloroacetic acid. After 10 min on ice, the denatured protein and cell debris were removed by centrifugation in a microcentrifuge for 10 min at 4°C. The thiol content of the supernatant solution was determined with 0.6 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) (16) in 0.2 M Na₃PO₄ buffer (pH 8.0). The concentration of DTNB derivatives of thiols was estimated spectrophotometrically at 412 nm. There were three replicates in each test, and the data are the means and SDs of three observations.

Detection of accumulation of reactive oxygen species.

Intracellular oxidant levels were determined by the use of 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Sigma), which is oxidized inside the cell to the fluorescent dichlorofluorescein (DCF). AmB-treated (0.125 μg/ml) sensitive and resistant parasites (2 × 10⁶ of each set) were incubated after the desired time intervals with 0.4 mM (final concentration) H₂DCFDA for 15 min in the dark. The cells were washed once in PBS (pH 7.2) and were harvested and lysed in lysis buffer (1% SDS and 1% Triton X-100 in 10 mM Tris), and the fluorescence intensity was measured in the supernatant using an LS55 spectrofluorimeter (Perkin Elmer), with excitation measured at 504 nm and emission at 529 nm. The measured fluorescence intensity was directly proportional to the accumulation of ROS. The reagent blank was prepared with 0.4 mM (final concentration) H₂DCFDA in lysis buffer. The accumulation of ROS has also been measured in the presence of the ROS scavenger 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Sigma). There were three replicates in each test, and the data are the means and SDs of three observations.

Inhibitor assay.

Verapamil, an ABC transporter inhibitor, BSO, an inhibitor of γ-GCS, and difluoromethylornithine (DFMO), an inhibitor of ODC, were added at concentrations of 5 μM each to study the effect of ABC transporters and thiol metabolic pathway inhibitors in strain reversal. They were added to the resistant parasites and incubated for 2 h at 23°C in a biological oxygen demand (BOD) incubator prior to AmB treatment. The parasites were subsequently washed with PBS (pH 7.2) and treated with AmB. Three experimental sets were prepared for the inhibitor assay: parasites preincubated with only verapamil, parasites pretreated with BSO and DFMO, and parasites pretreated with verapamil, BSO, and DFMO simultaneously. The LD₅₀ of AmB, intracellular thiol content, intracellular AmB content, and efflux of AmB in a drug-free medium were recorded. There were three replicates in each test, and the data are the means and SDs of these three observations.

Characterization of clinical isolates.

The parasites isolated from the splenic aspirates of VL patients were confirmed as Leishmania species by kDNA amplification (Fig. 1), and they were also demonstrated to be axenic, as they are clonally selected in NNN blood agar medium.

Confirmation of AmB-resistant and -sensitive nature of clinical isolates.

The LD₅₀ value for the resistant strain was found to be 8-fold higher than that of the sensitive strain, as demonstrated by both in vitro and ex vivo drug sensitivity assays (Table 1). We have also studied the cross-resistance property of AmB-resistant clinical isolates to structurally and functionally unrelated drugs and showed no significant cross-resistance toward other drugs, such as hexadecylphosphocholine, sodium antimony gluconate (SAG), paromomycin, and sodium stibogluconate (data not shown).

Increased membrane fluidity for the resistant strain.

The change in plasma membrane sterol content and composition must alter the physical state of the plasma membrane. DPH, a hydrophobic probe, shows a double hard-cone wobbling movement to incorporate a hydrocarbon inside the membrane bilayer, and orientation of the probe in the membrane is related to the physical state of the lipid bilayer (10). The emission anisotropy value of DPH fluorescence was used as a measure of membrane fluidity. Figure 2A demonstrated that the emission anisotropy value of AmB-resistant Leishmania promastigotes was ∼2.5 times lower than that of the sensitive promastigotes, indicating differences in the trimethylammonium (TMA)-DPH environment within the plasma membranes of these cells. The membrane of AmB-resistant cells is therefore more fluid than that of AmB-sensitive cells. This change in membrane fluidity is probably a consequence of the resistance-induced modification of membranous lipid metabolism.

Decreased K^+_i level for the sensitive strain.

The AmB-treated sensitive promastigotes demonstrated an ∼2-fold decrease in the K^+_i level compared to that of resistant promastigotes (Fig. 2B). The lower the K^+_i level is, the higher the K⁺ leakage will be and, consequently, the higher the membrane depolarization will be. Therefore, the membrane of the sensitive strain was more depolarized than that of the resistant strain after AmB treatment.

Expression pattern of SCMT gene.

SCMT (S-adenosyl-l-methionine:C-24-Δ-sterol methyltransferase) is an important enzyme in the sterol biosynthetic pathway, as it performs C-24 transmethylation, which is a key step in production of ergosterol. SCMT has two transcripts, SCMT A and SCMT B (48). We have found that SCMT A is absent in AmB-resistant parasites and expressed in sensitive parasites (data not shown), but the SCMT B was more highly expressed (∼2.5-fold higher) in the AmB-resistant parasites than in the AmB-sensitive parasites (Fig. 2C). These data have again been confirmed by quantitative real-time PCR (Fig. 2D). This result indicates an altered sterol biosynthesis in the resistant strain, which has a lower affinity for AmB.

Sterol analysis of sensitive and resistant parasites by GC/MS.

Sterol composition and content reported to be involved in the laboratory-derived AmB resistance of L. donovani parasites (38) were analyzed through GC/MS to understand sterol's involvement in conferring AmB resistance in clinical isolates of L. donovani. Recorded mass spectra (see the supplemental material) were used to identify the sterols (21, 22, 30, 37, 38). The fragment ions at m/z 458 (M⁺), m/z 468 (M⁺), m/z 454 (M⁺), and m/z 468 (M⁺) are indicative of cholesterol, ergosterol, cholesta-5,7,24-trien-3β-ol, and ergosta-5,7,24(28)-trien-3β-ol, respectively (see the supplemental material). GC/MS analysis showed that ergosterol (28%) and its isomer ergosta-5,7,24(28)-trien-3-β-ol (34%) are present in AmB-sensitive parasites, and these two components represent 62% of the total sterols in these parasites. In the case of AmB-resistant parasites, ergosterol is absent and the major sterol is cholesta-5,7,24-trien-3-β-ol (62%) (Table 2). Cholesterol is present in both parasites, as cholesterol is taken up from the culture medium, but it is not metabolized by the parasite in either AmB-resistant or -sensitive parasites. Mass spectra for the TMS ether derivatives are given in the supplemental material.

Expression levels of ABC transporters.

The relative expression levels of the ABC transporters reported to be involved in drug efflux/sequestration in Leishmania have been explored to understand the involvement of ABC transporters in conferring AmB resistance. The expression levels of MDR1 (homologous with the mammalian PgP cluster) (25) and PgPA (homologous with the mammalian MRP cluster) (45) were investigated by semiquantitative reverse transcriptase PCR (Fig. 3A) and further confirmed by real-time PCR (Fig. 3B). The mRNA level of MDR1 was approximately 4-fold higher in the resistant strain than in the sensitive strain, whereas the mRNA level of PgPA was about the same in both strains.

Intracellular AmB content.

The intracellular AmB content for drug-treated sensitive promastigotes was enhanced with an increase in incubation time, whereas that of the resistant strain increased slowly up to 8 h and decreased afterwards (Fig. 3C). It has also been observed that after 16 h of incubation, the intracellular AmB content of the sensitive promastigotes also starts to decrease significantly (Fig. 3C). We have also investigated the intracellular AmB content in the presence of verapamil, and the amount of intracellular AmB is lower than in uninhibited resistant parasites (Fig. 3C).

AmB efflux.

AmB efflux from the sensitive and resistant parasites has been studied by incubating the cells in a drug-free medium after treatment with the drug for specific time frames. It was observed that after 8 h of incubation with AmB, the resistant strain demonstrates an ∼2.9-fold-higher drug efflux than the sensitive strain (Fig. 3D).The AmB efflux for the resistant strain was also investigated in the presence of ABC transporter inhibitor verapamil. We found that the amount of AmB effluxed out in the drug-free medium is ∼2-fold less after 8 h for the resistant strain when preincubated with verapamil (Fig. 3D).

Expression levels of thiol metabolic pathway genes.

The expression levels of the important thiol metabolic pathway genes were investigated with late-log-phase cultures of AmB-treated resistant and sensitive parasites. The mRNA levels of the enzymes involved in biosynthesis of trypanothione and regulation of the reduced trypanothione level were found to be upregulated in the resistant strain compared to those in the sensitive strain. The expression levels of ODC, SPS, γ-GCS, TryS, and TR were found to be upregulated ∼2.3-fold, ∼2.2-fold, ∼2.8-fold, ∼2.2-fold, and ∼2.5-fold, respectively, in the resistant strain compared to the levels in the sensitive strain (Fig. 4). The gene of the tryparedoxin cascade (both cytosolic and mitochondrial) involved in peroxide elimination was also demonstrated to be upregulated in the resistant strain compared to its level in the sensitive strain. The mRNA levels of cTXN, CTP, mTXN, and MTP were found to be upregulated ∼2.7-fold, ∼2.3-fold, ∼3-fold, and ∼2.7-fold, respectively, in the resistant strain compared to the levels in the sensitive strain (Fig. 4). The above data were again validated by real-time PCR, and we observed similar variations in the expression levels of the investigated genes (Fig. 5A).

Intracellular reduced thiol levels.

The total intracellular reduced thiol content was measured using DTNB [5,5′-dithio-bis(2-nitrobenzoic acid)] and was found to be ∼2-fold higher in the resistant strain than in the sensitive strain (Fig. 5B). Total intracellular reduced thiol contents for both the resistant and sensitive strains had also been investigated in the presence of thiol metabolic pathway inhibitors BSO and DFMO. We found that the total reduced thiol content is decreased by ∼1.4-fold in the resistant strain when preincubated with both inhibitors (Fig. 5B). However, in the case of coinhibition with both thiol metabolic pathway and ABC transporter inhibitors, effects similar to those described above (data not shown) were found.

Accumulation of ROS.

The intracellular ROS production after AmB treatment was measured in a time-dependent manner using the nonfluorescent dye H₂DCFDA, which passively diffuses into cells, producing a polar diol by cleaving acetates of the dye using intracellular esterase, and gets retained inside the cells. The diol can then be oxidized by ROS to a fluorescent form, which can be measured for excitation at 480 nm and emission at 530 nm. The ROS levels in the resistant and sensitive strains were similar up to 16 h of incubation with the drug and were in the basal level (Fig. 5C). However, the ROS level for the sensitive strain started increasing steadily after 16 h and shot up to its maximum at 24 h and no significant change was observed in the resistant strain for the same time period (Fig. 5C). The ROS level of the sensitive strain was found to be ∼4-fold higher than that of the resistant strain at 24 h of incubation with the drug (Fig. 5C). Treatment with the ROS scavenger (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) significantly decreased the level of ROS in the sensitive strain (data not shown), and this, in turn, increased the rate of survival of the sensitive parasite, as the LD₅₀ increased ∼1.7-fold (Table 3). However, the ROS scavenger had no significant toxic effect on untreated resistant and sensitive parasites (data not shown).

Reversion of AmB resistance property by ABC transporter and thiol metabolic pathway inhibitors.

Verapamil, an inhibitor of MDR1, had no significant toxic effect on the untreated sensitive and resistant parasites (data not shown). Preincubation with verapamil demonstrated partial reversion of the resistant property, as the LD₅₀ of AmB for the resistant strain decreased ∼1.9-fold (Table 3). However, the LD₅₀ of the sensitive strain was not altered by preincubation with verapamil (the LD₅₀ decreased ∼1.01-fold). BSO and DFMO are inhibitors of γ-GCS and ODC, respectively, which are important rate-limiting enzymes of the thiol metabolic pathway. It has been demonstrated that BSO and DFMO have no significant toxic effect on the untreated sensitive and resistant parasites (data not shown). Preincubation with BSO and DFMO demonstrated a partial reversion of the resistant property, as the LD₅₀ of AmB for the resistant strain decreased ∼1.8-fold (Table 3). However, the LD₅₀ of the sensitive strain did not change significantly by preincubation with the same inhibitors (the LD₅₀ decreased ∼1-fold). Coinhibition with both thiol metabolic pathway and ABC transporter inhibitors demonstrated a much larger decrease in the LD₅₀ value (∼2.4-fold) for the resistant strain than did inhibition with either ABC transport inhibitors or thiol metabolic pathway inhibitors (Table 3), whereas the decrease in the LD₅₀ value was insignificant for the sensitive strain (∼1.04-fold) (Table 3). Therefore, coinhibition has a much more potent effect on the partial strain reversion.