Miltefosine-unresponsive Leishmania donovani has a greater ability than miltefosine-responsive L. donovani to resist reactive oxygen species

Authors


Abstract

Resistance of Leishmania parasites to miltefosine, which is only available oral drug, is a great concern. We have analyzed global gene expression profiles of miltefosine-unresponsive and miltefosine-responsive Leishmania donovani in order to understand the various metabolic processes involved in miltefosine drug resistance. The microarray data clearly indicated a role of oxidative metabolism in miltefosine resistance. Furthermore, fluorescence microscopy experiments suggested that miltefosine-unresponsive L. donovani resists the accumulation of reactive oxygen species and subsequent mitochondrial membrane damage leading to apoptotic death. In contrast, in miltefosine-responsive L. donovani, the accumulation of reactive oxygen species causes apoptotic death. Overall, this study provides fundamental insights into miltefosine resistance in L. donovani.

Database

The microarray data have been deposited in the Gene Expression Omnibus database under the accession number GSE45496

Abbreviations
APX

ascorbate peroxidase

FITC

fluorescein isothiocyanate

GO

gene ontology

LdFeSODA

Leishmania donovani mitochondrial iron superoxide dismutase-A

NAC

N-acetylcysteine

PI

propidium iodide

ROS

reactive oxygen species

SOD

superoxide dismutase

Introduction

The only available oral drug against for human visceral leishmaniasis is miltefosine (hexadecylphosphocholine), a lysophospholipid analog that has been suggested as a potential oral treatment for human visceral leishmaniasis [1]. Miltefosine was primarily developed as an anticancer drug, and it was found that the drug induces apoptosis in various cancer cell lines by targeting cell membranes [2, 3]. Subsequent studies have reported that miltefosine also causes apoptotic death of Leishmania parasites. The apoptotic death was mediated through inhibition of cytochrome c oxidase and mitochondrial dysfunction [4, 5]. It has been reported that Leishmania donovani mitochondrial iron superoxide dismutase-A (LdFeSODA) overexpression protects parasites from miltefosine [6]. Furthermore, prolonged incubation of parasites with miltefosine results in the release of LdFeSODA and cytochrome c, causing apoptotic death. It has been suggested that LdFeSODA protects the mitochondria of Leishmania from oxidative stress, thereby inhibiting programmed cell death [6]. However, there is no report on natural miltefosine resistance in relationship to reactive oxygen species (ROS). Furthermore, global gene expression profiles of miltefosine-unresponsive and miltefosine-responsive L. donovani strains have not been analyzed.

The miltefosine transporter LdMT and its specific β-subunit LdRos3 form the miltefosine translocation machinery at the Leishmania plasma membrane [7]. Miltefosine resistance in Leishmania is reported to be caused by single point mutations in these transporter proteins [8, 9]. The mutations in miltefosine transporters result in decreased uptake, increased efflux, and faster metabolism [7, 8]. It has also been reported that miltefosine resistance causes alterations in fatty acid and sterol biosynthesis (mainly Leishmania acyl-CoA synthetase and desaturase systems) that result in changes in the lipid composition of membranes of L. donovani promastigotes [10]. Changes in lipid composition in miltefosine-resistant L. donovani promastigotes alter membrane fluidity and permeability, and this may affect drug–membrane interactions [10]. However, the effect of metabolic changes in miltefosine-resistant L. donovani at the global gene expression level has not yet been studied. The aim of the current study was to analyze changes in the gene expression profile of miltefosine-unresponsive L. donovani with respect to miltefosine-responsive L. donovani. Data from gene expression profiling were experimentally validated. Our results indicate that miltefosine-unresponsive L. donovani accumulates ROS less than than miltefosine-responsive L. donovani. Apparently, miltefosine-unresponsive L. donovani has better redox machinery that can remove ROS more efficiently.

Results

Microarray data analysis

The data are plotted in the form of scatter plots and box whisker plots that show acceptable quality (Fig. 1). The data analysis indicated that 85 genes were upregulated and 42 genes were downregulated (Fig. S1). The upregulated and downregulated genes were annotated by use of the KEGG database (Table 1). The annotated upregulated genes are mainly involved in fatty acid metabolism, or encode proteins involved in oxidative metabolism (proteins of oxidative phosphorylation or enzymes involved in ubiquinone synthesis, a component of the electron transport chain). As alteration of fatty acid and sterol metabolism in miltefosine-unresponsive L. donovani has already been studied with biochemical methods [10], we focused primarily on oxidative metabolism. An efficient electron transport chain plays an important role in keeping the ROS concentration low [11]. Our microarray data (accession number GSE45496) suggests upregulation of genes involved in the synthesis of ubiquinone, an essential component of the electron transport chain, and a few other genes involved in oxidative phosphorylation.

Table 1. The pathways for the upregulated and downregulated genes were annotated by use of the KEGG database
KEGG ID pathwayNumber of geneGene symbol description
Pathways upregulated
ldo01100 Metabolic pathways4

LDBPK_010510 long-chain fatty acid CoA ligase, putative

LDBPK_280680 hypothetical protein

LDBPK_362740 folylpolyglutamate synthetase

LDBPK_365590 ubiquinone biosynthesis methyltransferase, putative

ldo00071 Fatty acid metabolism1LDBPK_010510 long-chain fatty acid CoA ligase, putative
ldo04146 Peroxisome1LDBPK_010510 long-chain fatty acid CoA ligase, putative
ldo00190 Oxidative phosphorylation2

LDBPK_181510 P-type H+-ATPase, putative

LDBPK_280680 hypothetical protein

ldo00790 Folate biosynthesis1LDBPK_362740 folylpolyglutamate synthetase
ldo00130 Ubiquinone and other terpenoid–quinone biosynthesis1LDBPK_365590 ubiquinone biosynthesis methyltransferase, putative
ldo04145 Phagosome1LDBPK_280680 hypothetical protein
ldo01110 Biosynthesis of secondary metabolites1LDBPK_365590 ubiquinone biosynthesis methyltransferase, putative
ldo03010 Ribosome1LDBPK_342620 40S ribosomal protein S19 protein, putative
ldo03008 Ribosome biogenesis in eukaryotes1LDBPK_301880 hypothetical protein
ldo03022 Basal transcription factors1LDBPK_301880 hypothetical protein
Pathways downregulated
ldo01100 Metabolic pathways2

LDBPK_151070 glutamate dehydrogenase

LDBPK_190710 glycosomal malate dehydrogenase

ldo00250 Alanine, aspartate and glutamate metabolism1LDBPK_151070 glutamate dehydrogenase
ldo00330 Arginine and proline metabolism1LDBPK_151070 glutamate dehydrogenase
ldo00630 Glyoxylate and dicarboxylate metabolism1LDBPK_190710 glycosomal malate dehydrogenase
ldo01110 Biosynthesis of secondary metabolites1LDBPK_190710 glycosomal malate dehydrogenase
ldo00020 Citrate cycle/tricarboxylic acid cycle1LDBPK_190710 glycosomal malate dehydrogenase
ldo00910 Nitrogen metabolism1LDBPK_151070 glutamate dehydrogenase
ldo03013 RNA transport1LDBPK_091130 eukaryotic translation initiation factor 2 subunit, putative
ldo00620 Pyruvate metabolism1LDBPK_190710 glycosomal malate dehydrogenase
Figure 1.

Analysis of microarray data (A) Scatter plot based on signal intensities: a graph of plotted points that show the relationship between two sets of data. The y-axis represents miltofesine resistance, and the x-axis indicates the control. A total of 85 genes were found to be upregulated, and 42 genes were downregulated. (B) Box whisker plot. Samples C1 and C2 represent two control experiments, and MR1 and MR2 represent two miltefosine-unresponsive experimental datasets.

Functional classification of gene expression data clearly indicated that genes involved in oxidoreductase activity and redox homeostasis are upregulated (Table 2). Taken together, the data indicated that miltefosine-unresponsive L. donovani has more efficient oxidative phosphorylation and a better ability to maintain redox homeostasis.

Table 2. Functional classification of gene expression data. The top five upregulated and downregulated GO terms were selected. The P-value represents the probability of obtaining the specified GO accession number from a list of random entities. The lower the P-value, the more significant is the GO accession number. Count in selection refers to the number of genes in the selected entity (for example, from t-test) list that have that particular GO term. Percentage count in selection refers to the percentage of genes in the input entity list that have that GO term. Count in total refers to the number of genes in all entities that have that GO term. Percentage count in total refers to the percentage of genes in all entities that have that GO term
GO IDGO Accession/GO function nameGO term/description of GOP-valueCount in selection% Count in selectionCount in total% Count in total
Upregulated functions
8424GO:0015036Disulfide oxidoreductase activity4.05 × 10−6515.15152270.781929
9708GO:0016667Oxidoreductase activity, acting on a sulfur group of donors1.54 × 10−5515.15152351.013611
20728GO:0045454|GO:0030503|GO:0045867|GO:0045868Cell redox homeostasis4.85 × 10−5515.15152441.274254
11673GO:0019725Cellular homeostasis5.42 × 10−5515.15152451.303215
18587GO:0042592Homeostatic process5.42 × 10−5515.15152451.303215
Downregulated functions
3809GO:0005509Calcium ion binding2.99 × 10−4421.05263621.79554
19582GO:0043648Dicarboxylic acid metabolic process7.88 × 10−4210.5263280.231683
2965GO:0004352Glutamate dehydrogenase activity5.50 × 10−315.26315810.02896
4270GO:00061032-Oxoglutarate metabolic process5.50 × 10−315.26315810.02896
4273GO:0006106Fumarate metabolic process5.50 × 10−315.26315810.02896

Flow cytometric studies

Parasites were treated with menadione (10 μm), a naphthoquinone, for 3 h to induce ROS, as these compounds have already been reported to induce ROS in the pathogen [6, 12], and flow cytometric analyses were performed. The data clearly indicated that miltefosine-responsive L. donovani (BHU-1081) generated more ROS than miltefosine-unresponsive L. donovani (BHU-1155) under similar menadione treatment. Furthermore, when miltefosine-responsive L. donovani was treated with miltefosine (25 μm), there was significant ROS generation, which is consistent with previous studies [13]. Interestingly, miltefosine-unresponsive L. donovani did not generate many ROS after similar treatment with miltefosine. In all cases where ROS were generated, pretreatment with N-acetylcysteine (NAC), a scavenger of ROS, was able to remove ROS, ruling out any experimental artefact (Fig. 2).

Figure 2.

Miltefosine-induced and menadione-induced ROS were measured by 2', 7'-dichlorofluorescein diacetate fluorescence, as assayed with flow cytometry. (A) Miltefosine-responsive L. donovani. (B) Miltefosine-unresponsive L. donovani. Control represents L. donovani promastigotes without any treatment; MF and ME represent miltefosine-treated and menadione-treated L. donovani promastigotes, respectively. NAC-MF and NAC-ME represent L. donovani promastigotes pretreated with NAC before miltefosine and menadione treatment, respectively. The data shown are representative of at least three experiments that gave similar results.

Apoptotic assays indicated that miltefosine-responsive L. donovani undergoes apoptosis [increased pro-pidium iodide (PI) and annexin V–fluorescein isothiocyanate (FITC) fluorescence] after menadione (10 μm) or miltefosine (25 μm) treatment. However, NAC pretreatment before menadione or miltefosine treatment prevented apoptosis, suggesting a clear link between ROS species and the apoptotic process. Additionally, when miltefosine-unresponsive L. donovani was treated similarly with menadione or miltefosine, no apoptosis was seen (Fig. 3).

Figure 3.

Flow cytometric analysis. L. donovani promastigotes treated with miltefosine or menadione for 6 h were double stained with PI and annexin V–FITC. Untreated L. donovani promastigotes after staining with PI and annexin V-FITC was used as control. MF and ME represent miltefosine-treated and menadione-treated L. donovani promastigotes, respectively. NAC-MF and NAC-ME represent L. donovani promastigotes pretreated with NAC before miltefosine or menadione treatment, respectively. The FL1 signal (x-axis) indicates the annexin V–FITC fluorescence value, and the FL2 signal (y-axis) represents the PI fluorescence value. The data shown are representative of at least three experiments that gave similar results.

Alteration in mitochondrial membrane potential

We observed a clear correlation between mitochondrial membrane damage, apoptosis and ROS generation in miltefosine-responsive L. donovani, more ROS being generated than in miltefosine-unresponsive L. donovani. After menadione (10 μm) or miltefosine (25 μm) treatment, miltefosine-responsive L. donovani showed disruption of mitochondrial membrane potential. NAC pretreatment before similar treatment with menadione or miltefosine in miltefosine-responsive L. donovani prevented disruption of mitochondrial membrane potential. Furthermore, menadione or miltefosine treatment in miltefosine-unresponsive L. donovani had no effect on mitochondrial membrane potential (Fig. 4).

Figure 4.

Miltefosine and menadione induce apoptosis as measured by mitochondrial membrane injury. L. donovani promastigotes were treated separately with IC50 doses of miltefosine and menadione for 6 h, and stained with a mitochondria-specific cationic dye, MitoCapture (Calbiochem). Untreated L. donovani promastigotes were used as controls. MF and ME represent miltefosine-treated and menadione-treated L. donovani promastigotes, respectively. Control, miltefosine-treated and menadione-treated cells were photographed with excitation wavelengths of ~ 570 nm (red) and ~ 500 nm (green), respectively, under a fluorescence microscope (× 60). Normal mitochondrial membrane potential is indicated by red fluorescence, and depolarization of the mitochondrial membrane is indicated by green fluorescence. The data shown are representative of at least three experiments that gave similar results.

Intracellular Ca2+ measurement

The increases in cytosolic Ca2+ concentration in miltefosine-responsive and miltefosine-unresponsive L. donovani promastigotes either treated or untreated with IC50 doses of miltefosine or menadione for 6 h are shown in Fig. 5. As shown, the measured fluorescence intensity at 510 nm in the miltefosine-responsive cells pretreated with IC50 doses of miltefosine or menadione increased, indicating the formation of a larger amount of Ca2+–MagFura complex. These data indicate disruption of mitochondrial membrane potential. Pretreatment with NAC, a scavenger of ROS, before miltefosine or menadione treatment led to a cytosolic Ca2+ concentration similar to that in controls (Fig. 6). Overall, the results suggest that the increase in Ca2+ concentration is linked to ROS accumulation. It is worth mentioning that miltefosine-unresponsive L. donovani showed less ROS accumulation and Ca2+ release than miltefosine-responsive L. donovani, and no mitochondrial membrane damage.

Figure 5.

Increase in cytosolic Ca2+ concentration in miltefosine-responsive and miltefosine-unresponsive L. donovani promastigotes separately treated with IC50 dose of miltefosine (MF) or menadione (ME) for 6 h and stained with Fura 2AM. Control data indicate L. donovani promastigotes without any treatment; NAC data indicate NAC pretreatment before miltefosine or menadione treatment. Fluorescence was measured with excitation and emission at 340 nm at 510 nm, respectively. Data represent the mean ± standard deviation of three independent experiments.

Figure 6.

SOD and APX assay. Miltefosine-unresponsive L. donovani showed higher SOD and APX activity than miltefosine-responsive L. donovani. Data represent the mean ± standard deviation of three independent experiments.

Redox enzyme assay

Miltefosine-unresponsive L. donovani showed higher superoxide dismutase (SOD) activity (466 unit mg·protein−1) and ascorbate peroxidase (APX) activity (2.6 mm·min−1 mg·protein−1) than miltefosine-responsive L. donovani (400 unit mg·protein−1 and 1.5 mm·min−1 mg protein−1, respectively) (Fig. 6). The increase in activity of redox enzymes in miltefosine-unresponsive L. donovani confirms that the mechanism of resistance against miltefosine is multifactorial, and that redox metabolism plays an important role. The data suggest that integrating redox modulators into conventional chemotherapy against leishmaniasis may be a viable strategy against miltefosine-resistance Leishmania isolates.

Discussion

Several studies on miltefosine-unresponsive Leishmania have been performed [7, 8, 10]. All studies support a role of fatty acid and steroid metabolism, as well as role of mutations in the miltefosine transporter LdMT. Although there has been a substantial increase in our knowledge relating to miltefosine resistance in Leishmania, there is still much that is not understood. The role of ROS in killing Leishmania parasites is well documented [14-16]. For the first time, we report experimental data that explain the relationship between parasite defense against ROS and Leishmania resistance to miltefosine. Our microarray data suggest that the metabolism of miltefosine-unresponsive Leishmania is better at resisting oxidative stress. The biochemical studies reported in the present article conclusively show that miltefosine-responsive Leishmania and miltefosine-unresponsive Leishmania promastigotes have different abilities to resist ROS. Our data indicate that miltefosine-unresponsive Leishmania accumulates a lower number of ROS under conditions that are sufficient to cause ROS-induced mitochondrial membrane potential damage and apoptotic death of miltefosine-responsive Leishmania. Additionally, we found higher SOD and APX activity in miltefosine-unresponsive Leishmania. SOD is a vital enzyme that detoxifies superoxide to H2O2 (less toxic than superoxide) and oxygen, and APX scavenges H2O2 and converts it into water. SOD and APX protect the parasite from oxidative stress and subsequent damage. There is a direct correlation between ROS and mitochondrial damage and change in mitochondrial membrane potential. The results conclusively show that miltefosine-unresponsive Leishmania has an improved ability to resist ROS.

Miltefosine and many other drugs against Leishmania cause redox imbalance in the parasite. The widely used antimony-based drugs have also been reported to act via important enzymes involved in redox homeostasis [17]. The mechanism of miltefosine resistance in Leishmania mediated through the redox system seems to be an adaptive change in the parasites resulting from the indiscriminate use of drugs.

Experimental procedures

Parasites, cell lines, and chemicals

Miltefosine-responsive L. donovani (BHU-1081) and miltefosine-unresponsive L. donovani (BHU-1155) were obtained from S. Sundar (Banaras Hindu University, India), and cultivated in M199 liquid medium supplemented with 15% heat-inactivated fetal bovine serum, 100 U·mL−1 penicillin, and 100 μg·mL−1 streptomycin. Miltefosine-unresponsive L. donovani was obtained from splenic material of a patient after a month of miltefosine treatment (18-year-old male with no past history of the disease). All chemicals used were of the highest grade, and were obtained from Sigma-Aldrich or Merck.

Microarray data collection and analysis

An L. donovani microarray (slides, 8 × 15k; Agilent MicroArray Design Identifier 035638) was used for analysis of miltefosine-responsive L. donovani and miltefosine-unresponsive L. donovani. Total RNA was isolated with the RNAeasy Mini Kit (Qiagen product ID 74104), accortding to the manufacturer's protocol, and the concentration and purity of the RNA extracted were evaluated spectrophotometerically. The integrity of the extracted RNA was confirmed with a bioanalyzer (Agilent; 2100). We considered RNA to be of good quality on the basis of the 260 nm/280 nm values, rRNA 28S/18S ratios, and RNA integrity number (RIN-Bioanalyzer).

The microarray experiment was performed at Genotypic Technology Private Limited (Bangalore, India), according to their optimized procedure. In brief, the samples were labeled by use of the Agilent Quick Amp Kit (Part number: 5190-0442). Five hundred nanograms of total RNA was reverse transcribed with oligodT primer tagged to the T7 promoter sequence. The cDNA thus obtained was converted to double-stranded cDNA in the same reaction. The cDNA was converted to cRNA in the in vitro transcription step, with T7 RNA polymerase, and Cy3 dye was added to the reaction mix. During cRNA synthesis, Cy3 dye was incorporated into the newly synthesized strands. The cRNA obtained was cleaned with Qiagen RNeasy columns (Qiagen; Cat. No. 74106). The concentration and amount of dye incorporated were determined with the nanodrop. technique. Samples that passed the quality control for specific activity were taken for hybridization. Six hundred nanograms of labeled cRNA was hybridized on the array with the Gene Expression Hybridization kit (Part No. 5190-0404; Agilent) in Sure hybridization Chambers (Agilent) at 65 °C for 16 h. Hybridized slides were washed with Agilent Gene Expression wash buffers (Part No. 5188-5327). The hybridized, washed microarray slides were then scanned on a G2565C scanner (Agilent Technologies). Images were quantified with feature extraction software (Version 10.7; Agilent). Feature-extracted raw data were analyzed with genespring gx Version 12.0 software from Agilent. Normalization of the data was performed in genespring gx, using the 75th percentile shift. (Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted. This normalization takes each column in an experiment independently, and computes the nth percentile of the expression values for this array, across all spots, where n has a range from 0 to 100, and n = 75 is the median. Significant genes that were upregulated and downregulated, showing one-fold and above increased expression within the samples with respect to control samples, were identified. A Student's t-test P-value was calculated for the replicate samples. Differentially regulated genes were clustered by using hierarchical clustering based on a Pearson coefficient correlation algorithm, to identify significant gene expression patterns. Functional classification was performed on the basis of gene ontology (GO) functions. Pathway analysis was performed with the KEGG database.

Generation of ROS

Parasites were treated with menadione (10 μm), a naphthoquinone, for 3 h to induce ROS, as these compounds have been reported to induce ROS in the pathogens.

Flow cytometric studies

Flow cytometric studies were carried out with a BD FACS Calibur flow cytometer, and analysis was performed with cellquestpro software (Becton Dickinson). ROS in L. donovani promastigotes (miltefosine-responsive and miltefosine-unresponsive) were measured with a cell-permeable probe, 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester, by flow cytometry, as reported previously [14, 15]. One of the hallmarks of apoptosis is exposure of phosphatidylserine on the cell surface, which can be detected by using FITC-conjugated annexin V. Necrotic cells also bind to annexin V–FITC, but are also stained with PI, as membrane damage in necrotic cells allows PI to enter the cell and stain DNA. The apoptosis in L. donovani was studied with an annexin V–FITC apoptosis detection kit (Calbiochem), with staining with annexin V–FITC antibody and PI according to the manufacturer's instructions. For all flow cytometric studies, cells were washed twice in NaCl/Pi before flow analysis.

Alteration in mitochondrial membrane potential

The mitochondrial transmembrane potential of the parasites was studied by fluorescence microscopy after staining with the MitoCapture apoptosis detection kit (Calbiochem). The kit utilizes MitoCapture, a cationic dye that fluoresces differently in healthy and apoptotic cells. In healthy cells, MitoCapture accumulates and aggregates in the mitochondria, giving off bright red fluorescence. In apoptotic cells, MitoCapture cannot aggregate in the mitochondria, owing to the altered mitochondrial transmembrane potential, and therefore remains in the cytoplasm in its monomeric form, fluorescing green. In brief, promastigote Leishmania cells (1 × 107 cells mL−1) were harvested by centrifugation at 1000 g for 5 min at 4 °C, and washed with cold NaCl/Pi. The cells (1 × 106 cells·mL−1) were suspended in 100 μL of incubation buffer containing MitoCapture reagent, which was diluted according to the manufacturer's instructions, and incubated at room temperature for 30 min (time optimized for good fluorescence image). After being stained, the cells were washed twice with NaCl/Pi, mounted on the glass slide, and photographed under a fluorescence microscope (Motic AE31).

Intracellular Ca2+ measurement

The fluorescent probe Fura 2AM was used to measure intracellular Ca2+ concentration [18]. Briefly, miltefosine-sensitive and miltefosine-insensitive cells that had been subjected to different treatments were harvested and washed twice (500 μL of buffer each time) with wash buffer containing 5.5 mm glucose, 116 mm NaCl, 0.8 mm MgCl2, 5.4 mm KCl, and 50 mm MOPS (pH 7.4). Cells were then incubated at 25 °C with 8 μm Fura 2AM in the same buffer containing 15% sucrose for 6 h. Cells were centrifuged (1000 g for 5 min), washed twice, and suspended in the same wash buffer. Fluorescence was measured with excitation and emission at 340 nm at 510 nm, respectively.

Redox enzyme assay

Equal numbers of Leishmania promastigote cells (both miltefosine-responsive and miltefosine-unresponsive) were taken separately, harvested by centrifugation (1000 g for 5 min), and washed twice with cold NaCl/Pi. The pellet was dissolved in 1 mL of 20 mm Tris/HCl (pH 7.5), protease cocktail inhibitors, and 1.5 mm MgCl2, and sonicated. After centrifugation, the supernatant was used for enzyme activity assay. SOD assay was performed with the riboflavin-mediated Nitro Blue tetrazolium reduction method [19]. The total 3-mL volume SOD reaction mixture included 13 mm methionine, 75 μm Nitro Blue tetrazolium, 0.1 mm EDTA and 4 μm riboflavin in 20 mm Tris/HCl (pH 7.5) in a glass test tube. All tubes were placed under fluorescent light (13 W) in an aluminum foil-lined box for 15 min, and spectrophotometric reading was performed at 560 nm. The blank and control were without illumination and without enzyme respectively. One unit is the amount of SOD that inhibits the rate of increase in absorbance resulting from Nitro Blue tetrazolium–diformazan formation by 50%.

APX assay was performed as described previously [20, 21]. APX assay mixture (1 mL) contains 500 μm ascorbate solution, 10 μm H2O2, 100 μm EDTA and 100 μL of Leishmania cell lysate in 12 mm Tris/HCl (pH 7.5). The oxidation of ascorbate was monitored by the decrease in absorbance at 290 nm in 3 min, and activity was calculated by the use of the extinction coefficient (2.8 mm−1·cm−1).

Acknowledgements

Research fellowships to M. Das and P. Saudagar from IIT Guwahati are acknowledged. Financial support by the Department of Biotechnology, Government of India in the form of research grants (Project nos. BT/01/IYBA/2009 and BT/PR3409/MED/29/326/2011) to V. K. Dubey is also acknowledged. M. Das and P. Saudagar performed the experiments and wrote the manuscript. S. Sundar and V. K. Dubey conceptualized the study and edited the manuscript. We acknowledge Genotypic Technology Private Limited Bangalore for the microarray processing.

Ancillary