JAC Advance Access published online on January 9, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl494
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Role of ABC transporter MRPA, 
-glutamylcysteine synthetase and ornithine decarboxylase in natural antimony-resistant isolates of Leishmania donovani
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110 067, India 2 Centre de Recherche en Infectologie du Centre De Recherche du CHUL and Department of Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada 3 Department of Pharmacology, Institute of Postgraduate Medical Education & Research, 244B Acharya JC Bose Road, Kolkata – 700 02, India
* Corresponding author. Tel: +91-11-26106630; Fax: +91-11-26165886; E-mail: madhubala{at}mail.jnu.ac.in
Received 23 August 2006; returned 27 October 2006; revised 6 November 2006; accepted 12 November 2006
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OBJECTIVES: The resistance of clinical isolates of Leishmania donovani to sodium antimony gluconate (SAG), the mainstay of treatment in Indian visceral leishmaniasis, has become a critical issue in India. The present work investigates the mechanism of resistance to SAG in parasites isolated from patients who are unresponsive to SAG.
METHODS AND RESULTS: Susceptibility to SAG as determined in vitro with intracellular amastigotes correlated well with the clinical response. The ABC transporter gene MRPA was amplified in resistant field isolates as part of an extrachromosomal circle. Co-amplification of the pterin reductase gene (PTR1) and MRPA suggests amplification of the H locus in SAG-resistant isolates. Amplification of MRPA was correlated to increased RNA as determined by real-time PCR. MRPA is an ABC-thiol transporter, and cysteine and glutathione were increased in the resistant isolates. Ornithine decarboxylase (a rate limiting enzyme in polyamine biosynthesis), and 
-glutamylcysteine synthetase (a rate limiting enzyme in glutathione biosynthesis), the two building blocks of the main cellular thiol trypanothione, were overexpressed in some of the resistant isolates.
CONCLUSIONS: A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region.
Key Words: drug resistance , sodium antimony gluconate , Leishmania donovani
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Introduction |
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Leishmaniasis is caused by a protozoan parasite that gives rise to a wide spectrum of diseases, ranging from the simple self-limiting cutaneous form to the debilitating visceral form, which is often fatal if left untreated. Although pentavalent antimonials like sodium antimony gluconate (SAG) are the age-old conventional therapy for visceral leishmaniasis (VL), in more recent times, increasing resistance to SAG has emerged as a major barrier in the treatment of VL.1 There has been an epidemic of primary resistance to antimonial drugs in parts of India1 and an urgent need exists to define the mechanism(s) of resistance.
The precise mechanism of action of SAG remains an enigma but it is generally agreed that pentavalent Sb(V) is reduced to trivalent antimony Sb(III), which constitutes the active form of the drug against the parasite.2 Reduction of the metal may take place either in the parasites3–6 or in the macrophages,7 or in both. To date, work on antimony resistance in Leishmania spp. has been mostly on laboratory mutants, in which resistance was induced in vitro in the presence of antimony or to the related metal arsenic.8–10 A consistent resistance mechanism deduced from in vitro work involves multiple steps where overproduction of ornithine decarboxylase (ODC), the rate limiting enzyme of the polyamine biosynthetic pathway11 and -glutamylcysteine synthetase (GCS), the rate limiting enzyme of glutathione (GSH) biosynthesis8,12 leads to overproduction of trypanothione [T(SH)2],8,10 which is the major reduced thiol of Leishmania and composed of a N1,N8-bisglutathione spermidine conjugate.13 Trypanothione is thought to bind to the active reduced form of the metal14 and these metal–trypanothione conjugates are either sequestered into an intracellular organelle by the ABC transporter, MRPA15 (formerly known as P-glycoprotein A, PGPA) or extruded outside the cell by an efflux pump.16 Resistance in field isolates may also involve amplification of genes other than the above-described metabolic pathway.17 Yet another mechanism leading to down-regulation of an uptake system is the loss of an aquaglyceroporin (AQP1) allele that has been reported to cause an increase in resistance to SAG.18,19
Resistance to SAG in field isolates is less well defined, and only recently were susceptibility values, determined using in vitro assays, found to correlate with the clinical response.20–22 We found that several of the findings elucidated while studying resistance in laboratory strains are also operational in field isolates. The mechanism(s) of resistance to SAG varied in isolates from the same geographical area. Understanding the mechanisms of antimony resistance in field isolates of Leishmania donovani will aid in development of strategies to avoid or overcome drug resistance.
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Materials and methods |
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Parasite and culture conditions
Promastigotes of Indian Leishmania donovani clones, GE1 (MHOM/IN/80/GE1F8R),23 AG83 (MHOM/IN/80/AG83) and four untyped clonal strains 2001, 41, NS2 and CK2 were isolated from patients with VL and were routinely cultured at 22°C in M-199 medium (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco/BRL, Life Technologies Scotland, UK) and 0.13 mg/mL penicillin and streptomycin.
Clinical isolates obtained prior to drug treatment from VL patients who had responded to SAG chemotherapy were designated as SAG-S (SAG-sensitive), whereas VL patients who did not respond to SAG were designated as SAG-R (SAG-resistant). Accordingly, SAG-S isolates used in this study include AG83-S and 2001-S, whereas the four SAG-R isolates were 41-R, GE1-R, NS2-R, and CK2-R. The SAG-R isolates were maintained in the absence of drug pressure in vitro. The isolates have been passaged through hamsters or BALB/c mice to retain their virulence, and importantly, their chemosensitivity profiles have remained unchanged as measured periodically by amastigote-macrophage infectivity assay described below.
DNA construct and transfection
An episomal Leishmania expression vector (pGL-
NEOLUC) containing luciferase-encoding DNA and a neomycin phosphotransferase selectable marker24 was used in the present study.
The construct (20 µg) was transfected into L. donovani promastigotes by electroporation in 2 mm gap cuvettes at 450 V, 500 µF (BTX Electro Cell Manipulator 600). Transfectants were selected for resistance to G418 (50 µg/mL) as described previously.25,26
Chemosensitivity profiles of SAG-S and SAG-R strains in a macrophage model
Stationary phase Leishmania promastigotes expressing the luciferase gene (pGL-NEOLUC) were used to infect J774A.1 macrophages. Macrophage cell line J774A.1 (American Type Culture Collection) was maintained at 37°C in RPMI-1640 medium (Sigma) containing 10% heat inactivated FBS as described previously.27 Briefly, J774A.1 murine macrophages (1 x 105 cells/250 µL/well) were infected with 1 x 106 promastigotes in M199 media with 10% FBS. After 3 h, the non-internalized parasites were washed off and SAG was added at different concentrations (10–100 µg/mL). After 5 days of drug exposure, plates containing adherent macrophages were washed and luciferase activity was determined.24 The 50% inhibitory concentration (IC50) was determined from the graph representing different concentrations of SAG plotted against relative light units (RLU) produced by luciferase-expressing parasites.
Nucleic acid isolation, PFGE and hybridization analysis
Genomic DNA was isolated from 
2 x 109 promastigotes by a standard procedure,28 and circular DNA was isolated by Promega Wizard miniprep kit following the manufacturer's instructions. Genomic DNA and circular DNA were digested with HindIII and subjected to electrophoresis in 1% agarose gels. The fragments were transferred to HybondTM–N+membrane (Amersham Pharmacia Biotech) and subjected to Southern-blot analysis.
Total RNA was isolated from 2 x 108 promastigotes using TRI ReagentTM (Sigma). For northern-blot analysis, 15 µg of total RNA was fractionated by denaturing agarose gel electrophoresis and transferred onto nylon membrane following standard procedures.
Chromosomes of the clinical isolates were separated by PFGE in which low melting agarose blocks, containing embedded cells (108 log phase promastigotes per mL) were electrophoresed in a contour clamped homogeneous electric field apparatus (CHEF DRIII, Bio-Rad) in 0.5 x Tris-borate-EDTA, with buffer circulation at a constant temperature of 14°C and run time of 24 h, using Saccharomyces cerevisiae chromosomes as size markers.
Following the transfer of DNA, RNA and chromosomes on to nylon membranes, the nucleic acids were cross-linked to the membrane with ultraviolet light in a Stratagene UV cross-linker. The blots were hybridized with denatured [-32P]dCTP-labelled DNA probe. The DNA probes used in the present study included a 400 bp MRPA fragment (released from plasmid PM12 that was digested with BamHI and PstI), a 2.3 kb fragment GSH1 probe (derived from plasmid psphygro-GCS digested with HindIII and XbaI), a 2 kb ODC full-length probe (derived from plasmid psphygro-L. donovani ODC by digesting with HindIII and XbaI) and a 1.6 kb 5'-PTR1 probe (derived from plasmid psp72-Y-hygro-5'-PTR1).
The human leukaemia cell line THP-1 was infected with promastigotes of L. donovani at a ratio of 15 : 1 as described earlier21,29 and RNAs from intracellular parasites were obtained as described by Decuypere et al.30 Quality and quantity of the RNA were determined using the RNA 6000 NanoLabchip kit on the Bioanalyzer 2100 (Agilent Technologies). The sequences of the primers for MRPA are forward 5'-GCGCAGCCGTTTGTGCTTGTGG and reverse 5'-TTGCCGTACGTCGCGATGGTGC, and for the GAPDH control forward 5'-GAAGTACACGGTGGAGGCTG and reverse 5'-CGCTGATCACGACCTTCTTC primers. Complementary DNAs from intracellular Leishmania were synthesized from 40 ng of total RNA using the SuperscriptIITM RNase H– Reverse Transcriptase (Invitrogen) and Oligo (dT)12–18 primers (Invitrogen) following manufacturer's instructions. Real-time PCR was performed in triplicate in 25 µL volumes using IQ SybrGreen Super mix (Bio-Rad) for detection in a Rotor Gene-3000 (Corbett Research). Reactions were run using the following thermal profile: initial denaturation at 95°C for 4 min followed by 40 cycles with denaturation at 95°C for 20 s, annealing at 62°C for 20 s and extension at 72°C for 20 s. The PCR was followed by a melt curve analysis to ascertain that the expected products were amplified. The relative amount of PCR products generated from each primer set was determined based on the threshold cycle (Ct) value and amplification efficiencies, and was normalized by dividing the values by the relative amount of the GAPDH gene used as a control.
Thiols were derivatized with monobromobimane and separated by high-performance liquid chromatography (HPLC) as described elsewhere.10
Late log phase promastigotes (1 x 108) were harvested and the resultant cell pellet was resuspended in lysis buffer (20 mM MOPS, pH 7.2; 1 mM dithiothreitol; 2 mM phenylmethylsuphonylfluoride; 0.5 µg/mL each of leupeptin and aprotinin).
The cell pellet was lysed by sonication and cell supernatants were prepared by centrifugation at 20 000 g. Protein (50 µg) of promastigotes was fractionated by SDS–PAGE, blotted on to PVDF membranes using electrophoretic transfer cell (Bio-Rad) and probed with ODC antibody (diluted 1 : 3000, a gift from Dr Buddy Ullman, Oregon, USA). Western-blot analysis was done using the ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol. The results shown are from a single experiment typical of at least three giving identical results.
Data were statistically analysed by the Student's t-test. The data represent mean ± standard deviation (SD) of three determinations from at least two independent experiments. A P value of <0.05 was considered significant.
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Characterization of the resistance phenotype in clinical isolates
PFGE of the chromosomes of L. donovani clinical isolates derived from patients responding or not to standard SAG treatment indicated a similar karyotype, as no major differences were observed in chromosome number and sizes (results not shown). The sensitivity of these related clinical isolates to SAG was tested in intracellular amastigotes. The Leishmania expression vector (pGL-NEOLUC) containing the luciferase gene was transfected into the field isolates and the resulting transfectants were used for susceptibility assays (Figure 1). The IC50 values of AG83-S and 2001-S coming from SAG-responsive patients were 9 ± 0.5 µg/mL and 13 ± 1.5 µg/mL, respectively, whereas the field isolates 41-R, GE1-R, NS2-R and CK2-R coming from SAG-unresponsive patients had IC50s of 65 ± 3.4 µg/mL, >100 µg/mL, 24 ± 1.4 µg/mL and 55 ± 2.5 µg/mL, respectively, as extrapolated from the predicted graphs.
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Role of ABC transporter MRPA in antimony-resistant L. donovani clinical isolates
Gene amplification is a hallmark for drug resistance in Leishmania, at least when resistance is induced in vitro.31–33 Gene amplification has also been noted in field isolates.17,33 Previous reports on antimonial resistance have shown an increased expression of an ABC transporter MRPA.8,29,34 To test whether MRPA was amplified in our field isolates, we prepared total DNA of field strains and hybridized the DNA to an MRPA-specific probe. Amplification of the MRPA gene was observed in three resistant isolates, GE1-R, NS2-R and CK2-R (Figure 2a). However, 41-R, another SAG-R isolate did not show any MRPA amplification (Figure 2a). The amplicons present in the resistant isolates were further analysed by PFGE. Amplicons found in the resistant isolates GE1-R, NS2-R and CK2-R were circular, as indicated by their characteristic migration in PFGE (Figure 2b) with the smears possibly corresponding to various topoisomers of the circles.35 The circular nature of amplicons in resistant Leishmania isolates was further confirmed by isolating them by standard plasmid alkaline lysis preparation and migration in agarose gel. Comparison of DNAs derived from sensitive and resistant field isolates revealed circular amplified band in the resistant isolates GE1-R, NS2-R and CK2-R that were absent in the sensitive 2001-S and AG83-S and also in the 41-R isolates (Figure 3a). These circular amplicons from GE1-R, NS2-R and CK2-R were isolated and digested with HindIII. A similar digestion pattern was observed on the ethidium-bromide-stained gel (Figure 3b). Southern-blot analysis of the ethidium-bromide-stained gel with an MRPA-specific probe showed the presence of MRPA on the circular amplicon (Figure 3b). MRPA is part of the H locus, a region that also contains the pterin reductase gene (PTR1).36,37 The PTR1 gene was indeed found to be co-amplified with MRPA as determined by Southern-blot analysis (Figure 3b).
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Customized DNA microarrays containing all ABC protein genes of Leishmania were hybridized to GE1-R and 2001-S labelled cDNAs. MRPA was also found to be consistently overexpressed in GE1-R (P < 0.01) (A. Mukherjee, M. Ouellette and R. Madhubala, unpublished observations). Clinical resistance is found in the intracellular parasites while the molecular mutations described here were studied with the promastigotes. While it is unlikely that genes amplified in promastigotes would not lead to increase RNA in intracellular parasites, we carried out a real time RT-PCR assay of MRPA using intracellular amastigotes infecting the THP-1 cell line. An example of real time RT-PCR is shown in Figure 4(a). Increased expression of MRPA was indeed noted in GE1-R, NS2-R, and CK2-R but not in 41-R or the sensitive 2001-S lines (Figure 4b).
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Role of
-glutamylcysteine synthetase and ornithine decarboxylase in antimony resistance in the clinical isolates
In addition to MRPA, several loci have been reported to be amplified in antimony-resistant isolates, one being the GSH1 gene coding for the heavy subunit of GCS, the rate limiting enzyme for GSH synthesis.12 Overexpression of ODC, the rate-limiting enzyme in polyamine biosynthesis, has also been reported in metal-resistant mutants.11 The parasite-specific spermidine–GSH conjugate, trypanothione [T(SH)2],13 is known to bind to antimony, and it is proposed that the Sb-T(SH)2 complex is either extruded outside the cell by an ATP-dependent efflux system16 or sequestered within a vacuole by MRPA.15 This prompted us to test whether ODC or GSH1 was overexpressed in our field isolates resistant to SAG. Southern-blot analysis showed no gene amplification of GSH1 (Figure 5a) although in the resistant strain 41-R, northern-blot hybridization with GSH1 showed an increased expression of two transcripts of 2.4 and 3.4 kb in size when compared with the other isolates (Figure 5b). Southern-blot analysis of total genomic DNA digested with HindIII and hybridized with a full-length ODC probe showed an amplification of the ODC gene in 41-R, GE1-R, NS2-R and CK2-R as compared with the sensitive strains AG83-S and 2001-S (Figure 5c). Western-blot analysis further showed an increased expression of ODC protein (Figure 5d).
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Thiol analysis in the clinical isolates
We also quantified thiol levels in the SAG-S and SAG-R isolates (Figure 6). Surprisingly, there were no differences in the T(SH)2 levels between the SAG-S and SAG-R isolates (Figure 6c). However, the levels of cysteine were increased by 2-fold in the SAG-resistant strains GE1-R, NS2-R, and 2.8-fold in CK2-R when compared with the 2001-S isolate. The increase in cysteine level in 41-R was 3.5-fold when compared with 2001-S (Figure 6a). Similarly GSH levels showed 2.8-, 1.8-, 2.0- and 1.75-fold increases in 41-R, GE1-R, NS2-R and CK2-R, respectively, compared with 2001-S (Figure 6b). The levels of thiols were not significantly different between 2001-S and AG83-S.
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0.04–0.016; **P = 0.006. (b) *P = 0.01; **P = 0.007–0.005; ***P = 0.001. (c) Not significantly different. |
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SAG is the drug of choice against Leishmania and resistance to this drug is a major problem in the field not only in the Indian subcontinent, but also throughout the world.1,19–21,38–40 This increase in resistance to SAG has led to an upsurge in therapeutic failure, and with the limited chemotherapeutic alternatives, it is extremely relevant that mechanisms of resistance be evaluated in field isolates.1
Gene amplification, at least in strains made resistant under laboratory conditions, is a frequent mechanism by which Leishmania resists the action of cytotoxic drugs.31–33,41 The first amplified gene in Leishmania promastigotes selected for arsenite resistance34 or Sb(III) resistance8 is the ABC transporter gene MRPA, which causes drug sequestration. Amplification of the ABC transporter gene MRPA has also been reported in an Sb(V) mutant of a Leishmania guyanensis cell line42 or in axenic amastigotes of Leishmania infantum selected for resistance to Sb(III).29 However, to date it remains an open-ended and unanswered question as to whether similar mechanisms exist in clinical isolates. To address this question we have characterized clinical isolates from India and report that linked but diverse mechanisms of resistance are operative in these isolates.
The ABC transporter MRPA has been shown to confer resistance to antimonials in promastigotes of L. tarentolae by sequestration of the metal thiol conjugates in an intracellular organelle located close to the flagellar pocket.15 This model also appears to be applicable to the amastigotes.29 For the first time we show that MRPA gene amplification is also taking place in some field isolates (Figure 2). MRPA is part of an extrachromosomal circle in GE-1-R, NS2-R, and CK2-R. This circular amplicon also encodes the pterin reductase gene (PTR1). Pterins are essential co-factors for Leishmania growth,43,44 and co-amplification of PTR1 may provide a growth advantage to resistant isolates under a number of conditions.
Previous studies using in vitro laboratory-generated drug-resistant Leishmania have shown that resistance to metals in Leishmania is multifactorial, with contributions by several independent mechanisms as mentioned earlier. In our study the ODC gene was amplified in the resistant isolates, 41-R, GE1-R, NS2-R and CK2-R. However, the ODC gene was not amplified on the extrachromosomal circles (data not shown). Western-blot analysis showed an increased expression of ODC protein in all the resistant isolates studied. Overexpression of ODC and GSH1 is usually linked to an increase, respectively, of polyamine or thiol levels, which can favour an increase in T(SH)2 levels.11 An increase in cysteine and GSH levels was observed (Figure 6) but we did not find any increase in trypanothione levels in any of the resistant isolates. This was also the case in L. infantum resistant to Sb(III).29 This is possibly because the levels of T(SH)2 in the L. tarentolae strain studied are much lower than in other cells. An alternative, non-exclusive, explanation is that the mode of action of Sb(V) was suggested to deplete T(SH)2 by efflux of Sb-trypanothione conjugate.14 Possibly this efflux system is increased in the resistant field strain, hence leading to increased trypanothione efflux and thus explaining a relatively constant level of trypanothione, despite that two biosynthetic steps are increased.
GSH1 was not amplified in any mutants but RNA overexpression was detected in 41-R. Overexpression without gene amplification has been observed in Leishmania-resistant cells11 and it is possible that an increased RNA stability is responsible for the augmented levels of RNA. The increase in cysteine or GSH could possibly have a role in SAG resistance by binding either directly or indirectly to the metal compound. In our study we also observed that 41-R, the only strain that overexpressed GSH1, had much higher levels of cysteine and GSH when compared with GE1-R, NS2-R and CK2-R. It remains to be determined how cysteine and GSH are increased in field isolates that do not overexpress GSH1. It is possible that other thiol biosynthesis genes, as shown for in vitro resistant isolates,25,29 are increased. However, the results of this study differ from a recent finding from studies on strains derived from Nepal,30 raising the possibility that different resistance mechanisms are operating in neighbouring countries.
This is the first demonstration that resistance mechanisms found in laboratory strains can also be found in clinical kala azar L. donovani isolates. Increased thiols and MRPA overexpression are shown here to be also important resistance factors in the field. As clearly shown from the limited strains studied, the expression of a number of different genes appears to be implicated in SAG resistance. Further work is warranted in a larger number of isolates to test the frequency of the mutations detected and find possibly other resistance determinants.
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None to declare.
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Acknowledgements |
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The work carried out in this paper was supported by a grant from the Council of Scientific and Industrial Research, Government of India, New Delhi, India to R. M. Both M. O. and R. M. were supported by a grant from the Canadian Institute of Health Research, Canada. A Senior Research Fellowship from the Council of Scientific and Industrial Research, India supported A. M., and K. P. is supported by a grant from the University Grants Commission, New Delhi, India. Work in M. O.'s lab is supported by CIHR operating and group grants. M. O. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology and a Canada Research Chair in Antimicrobial Resistance. M. C. gratefully acknowledges the grant support from the Council of Scientific and Industrial Research, Government of India, New Delhi and Dr S. Duttagupta and Dr S. Roy, Indian Institute of Chemical Biology, Kolkata, and also Dr N. Singh, Central Drug Research Institute, Lucknow for providing the strains for this work.
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