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International Journal of Antimicrobial Agents 36 (2010) 50–57

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

journa l homepage: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

verexpression of histone H2A modulates drug susceptibility in Leishmaniaarasites

uchi Singha,b,1, Dhiraj Kumara, Robert C. Duncanb, Hira L. Nakhasib, Poonam Salotraa,∗

Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, IndiaDivision of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, CBER, FDA, Bethesda, MD, USA

r t i c l e i n f o

rticle history:eceived 3 October 2009ccepted 3 March 2010

eywords:isceral leishmaniasiseishmaniaicroarrayistones

a b s t r a c t

Resistance to antimonials has emerged as a major hurdle to the treatment and control of visceral leish-maniasis (VL), also know as kala-azar (KA), the disease caused by Leishmania donovani, in India where>60% of KA patients are unresponsive to sodium antimony gluconate (SAG) treatment. Determinants ofresistance in laboratory strains are partly known, however the mechanism operating in field isolates isnot well understood. In microarray-based expression profiling with RNA isolated from field isolates ofdrug-resistant and -sensitive L. donovani parasites, genes encoding histone 1 (H1), histone 2A (H2A),histone 4 (H4), mitogen-activated protein kinase 1 (MAPK1) and two hypothetical proteins showedsignificantly higher expression in antimony-resistant parasites, whilst genes encoding an amino acid

odium antimony gluconaterug resistance

transporter showed higher expression in sensitive parasites. The expression level of these genes was val-idated by semiquantitative polymerase chain reaction (PCR). Furthermore, the higher expression of H1,H2A and MAPK1 was confirmed at the protein level in resistant isolates. Overexpression of H2A in a drug-sensitive laboratory strain as well as a field isolate of L. donovani resulted in conversion of SAG-sensitiveLeishmania parasites into a resistant phenotype. Moreover, H2A overexpression resulted in a significantdecrease in susceptibility towards other antileishmanial drugs currently in use, i.e. amphotericin B and

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. Introduction

Leishmaniases are endemic in 88 countries on five continentsAfrica, Asia, Europe, North America and South America), with aotal of 350 million people at risk [1]. Leishmania-endemic regionsave expanded significantly, accompanied by a sharp increase inhe number of recorded cases of the disease. Visceral leishmaniasisVL), also known as kala-azar (KA), is the most severe form of theisease that, if untreated, has a mortality rate of almost 100% andas an annual incidence of 500 000 cases [2].

Chemotherapy is critically important in reducing the burden ofisease, and pentavalent antimonials (SbV) are the first-line drugsor all clinical forms. Treatment failure is well documented for SbV,articularly in India where >60% of VL-patients are unresponsiveo SbV treatment and respond only to amphotericin B (AmB) [3].

nresponsiveness to SbV may be due to several factors of para-

ite or host origin, including the host immune response. There areow strong indications that treatment failure may be partly due tohe intrinsic drug resistance of the parasite [4–6]. The prevalence of

∗ Corresponding author. Tel.: +91 11 2616 6124; fax: +91 11 2616 6124.E-mail address: salotra@vsnl.com (P. Salotra).

1 1 Present address: National Institute of Malaria Research (ICMR), Delhi, India.

924-8579/$ – see front matter © 2010 Elsevier B.V. and the International Society of Chemoi:10.1016/j.ijantimicag.2010.03.012

in drug resistance.r B.V. and the International Society of Chemotherapy. All rights reserved.

drug resistance in such high proportions is unique to India, howevera varying proportion of antimony-resistant Leishmania parasiteshave been observed in several endemic regions such as Iran, Peruand Columbia [7–9].

Drug resistance is a complex phenomenon in the Leishmania par-asite, as several metabolic pathways and membrane transportersare implicated in the resistance phenotype. Different mechanismsfor drug resistance have been suggested, such as gene amplificationas well as the parasite’s inability to convert SbV to SbIII. It is agreedthat the trivalent antimony SbIII is the active form of the drug andis generated by reduction of SbV by thiols either by the parasites orby macrophages, or both [10,11]. A targeted DNA microarray with44 known genes known to be responsible for resistance has beenused to show the linkage of the genes to drug resistance in theLeishmania parasite [12].

Microarray technology and proteomic screeening have beenemployed to identify novel drug targets to overcome this situationand have provided a more rapid and high-throughput alternativefor elucidation of the mechanisms leading to resistance [13–16].

Various molecules such as multidrug resistance protein (MRPA),HSP83, nucleoside transporter, long-chain fatty acid–CoA ligase, asmall kinetoplastid calpain-related protein, along with many hypo-thetical proteins [15,16] have been identified using these methods.Recently, using transcriptome profiling, differentially expressed

otherapy. All rights reserved.

l of Antimicrobial Agents 36 (2010) 50–57 51

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Table 1Gene-specific primers used in reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR.

Gene Primer sequence Amplicon size (bp)

Histone 1 F, 5′-CTC GCC GCA GAA GTC TCC-3′ 206R, 5′-TTC TTC GCC GAG GAT TTC-3′

Histone H2A F, 5′-CAG CCG TGC TGG AGT ACC TG-3′ 233R, 5′-TGT CGC TTG CCC TTC TTG-3′

Histone H4 F, 5′-ATG GCC AAG GGC AAG CGC CT-3′ 246R, 5′-CGC CGT CAC CGT CTT CTT G-3′

MAPK F, 5′-GGT GTG TGA TTG GGG AGA TGC-3′ 219R, 5′-TCG CCT CGC TAT CCT TCA GG-3′

58A8 F, 5′-AGC ACA TGC AGG AGC TGT GG-3′ 228R, 5′-CTT GAA CTC AGC GTA CTG CGG-3′

87B9 F, 5′-GAT TAC TGG GGC GAC AAC TAC G-3′ 193R, 5′-TCT TCA GCG GCT CGT GAC C-3′

42G8 F, 5′-AAC GCC TCT GGT CTT GTT ATG-3′ 236R, 5′-GCC GTC AGC ACA TCC TTC AC-3′

MRPA F, 5′-GCG CAG CCG TTT GTG CTT GTG G-3′ 179R, 5′-TTG CCG TAC GTC GCG ATG GTG C-3′

GSH1 F, 5′-CAT TGG CTG GCG CGT TGA GTT C-3′ 166R, 5′-ATG TGC GCG GCC CAT ATT CTC G-3′

AQP1 F, 5′-TTT GGA ACC GGC GTC GTT GC-3′ 182R, 5′-ACA CAG TTC GCC AGC GTT ACG G-3′

R. Singh et al. / International Journa

enes in antimony-resistant Leishmania infantum were found to behysically linked in the genome, and the correlation of antimonyesistance levels and the copy number of aneuploid chromosomesuggest a putative link between aneuploidy and drug resistance16]. Available information on antimony resistance indicates thateveral mechanisms may coexist in the same cell and that dif-erent mechanisms may operate in field isolates compared withaboratory-generated resistant parasites [11,17]. In this study, wexploited Leishmania donovani genomic microarray for evaluationf parasite gene expression between antimony-sensitive versusesistant strains isolated from VL patients and identified variousenes, primarily histones H1, H2A and H4 along with mitogen-ctivated protein kinases (MAPKs) and a few hypothetical proteins,hat were upregulated in resistant parasites.

. Materials and methods

.1. Materials

Sodium antimony gluconate (SAG) (Albert David Ltd., Kolkata,ndia), miltefosine (MLF) (Cayman Chemical Company, Annrbor, MI), AmB, potassium antimony tartrate (SbIII), RPMI640, M199 medium, anti-rabbit immunoglobulin G–horseradisheroxidase (IgG-HRP), anti-mouse IgG-HRP, anti �-tubulin andnti-haemagglutinin (HA) antibody (all from Sigma-Aldrich, Stouis, MO), BioMax MR X-ray film (Kodak, Rochester, NY), fetalovine serum (FBS) (Gibco, Grand Island, NY), 8-well chamberlides (Nunc, Rochester, NY), Diff-Quik® stain solution (Dadeehring, Newark, DE), TRIzol reagent, pZerO vector, SuperScriptTM

I RNase H-Reverse Transcriptase and Oligo(dT)12–18 (all from Invit-ogen, Carlsbad, CA), DNase I (Fermentas, Glen Burnie, MD), RNeasyolumns (QIAGEN, Valencia, CA), SYBR Green PCR Master MixApplied Biosystems, Foster City, CA), nitrocellulose membranesnd Western Blot Enhanced Chemiluminescence (ECL) Detectioneagent (Millipore, Billerica, MA) were employed for the study.

.2. Parasite culture and generation of antimony-resistanteishmania donovani (K80SbIII)

Three clinical isolates obtained from either SAG-unresponsiveK80 (MHOM/IN/1999/K80)] or SAG-responsive [K133MHOM/IN/2000/K133) and K135 (MHOM/IN/2000/K135)]atients were used in the present study [6]. Cryopreservedarasites were used for experimental work within six passagesfter isolation from the patients. Reference Leishmania isolates usedn this study were L. donovani Sudan [LdS (MHOM/SD/62/1S-C12D]nd L. donovani AG83 [LdAG83 (MHOM/IN/83/AG83)]. Promastig-tes were maintained in M199 medium supplemented with 10%BS at 26 ◦C. K80 was adapted as an SbIII-resistant parasite byn vitro passages with a stepwise increase in the concentrationf SbIII from 10 �g/mL to 125 �g/mL (K80SbIII) in the M199edium + 10% FBS. The drug concentration was increased onlyhen the drug-exposed parasites showed a growth rate equivalent

o that of the parallel-growing wild-type parasites.

.3. In vitro assay for drug sensitivity

Parasite isolates were analysed for in vitro drug susceptibilitys described previously [5]. Briefly, the mouse macrophage adher-nt cell line J774A.1 (2 × 105 cells/well) in 8-well chamber slidesas infected with stationary-stage promastigotes at a 10:1 (L. dono-

ani:macrophage) ratio and was incubated in 5% CO2 for 4 h at 37 ◦C.fter washing, the cells were incubated for 12–18 h. Cells weree-incubated for 48 h with SAG (0, 3, 10, 30, 60 and 100 �g/mL),LF (0, 0.5, 1.25, 2.5, 5, 10 and 30 �g/mL) or AmB (0, 0.25, 0.5, 1.0,

.0, 3.0 and 4.0 �g/mL). After staining with Diff-Quik solutions, the

GADPH F, 5′-GAA GTA CAC GGT GGA GGC TG-3′ 206R, 5′-CGC TGA TCA CGA CCT TCT TC-3′

MAPK, mitogen-activated protein kinase.

numbers of amastigotes per cell were counted in 100 macrophages.Percent killing was calculated by sigmoidal regression analysis (Ori-gin 6.0; OriginLab Corp.).

2.4. RNA isolation and real-time polymerase chain reaction (PCR)analysis

Total RNA was prepared using TRIzol reagent from mid log-phase Leishmania. RNA was treated with RNase-free DNase I toavoid any genomic contamination and was further purified usingRNeasy columns. Complementary DNA was re-synthesised from5 �g of total RNA using SuperScriptTM II RNase H-Reverse Tran-scriptase and Oligo(dT)12–18 primers following the manufacturer’sinstructions. Real-time PCR was performed in triplicate in 25 �Lvolumes using SYBR Green PCR Master Mix in an ABI Prism 7000Sequence Detection System (Applied Biosystems). The relativeamount of PCR product generated from each primer set was deter-mined based on the threshold cycle (Ct) value and was normalisedby the relative amount of GAPDH gene used as a control. Quan-titative expression was calculated with respect to the referencesensitive isolate LdAG83. Various primers used in the study aregiven in Table 1.

2.5. Transcriptome profiling of resistant and sensitive parasites

Transcriptome analysis was carried out as described elsewhere[18] using RNA isolated from K135 and K80SbIII. Briefly, a libraryof 1–1.5 kb randomly sheared fragments of genomic DNA from afresh isolate of L. donovani prepared from an Indian KA patient wasligated into pZerO vector. A total of 8448 PCR-amplified insertsfrom library clones, representing ca. 60% of the expressed genes,along with alien external DNA, 24 known Leishmania genes and 12negative controls were printed in duplicate on poly-lysine slides.

The quality and quantity of purified RNA were assessed usingRNA 6000 Nano Assay Chips on a Bioanalyser 2100 (Agilent Tech-nologies, Santa Clara, CA). To minimise variations in RNA quality,

cells were collected at identical growth points under identical con-ditions. Fluorescently labelled cDNA was synthesised from totalRNA of drug-sensitive and -resistant parasites as described pre-viously [18]. The two fluorescently labelled cDNAs were mixedtogether with Ambion hybridisation buffer and hybridised with the

5 of Antimicrobial Agents 36 (2010) 50–57

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Fig. 1. Characterisation of Leishmania donovani isolates. (A) Susceptibility of L. dono-vani isolates K80SbIII (resistant) and K135 (sensitive) to sodium antimony gluconate(SAG) measured by intracellular assay in J774 macrophage cells. Each data point rep-resents the mean ± standard deviation (S.D.) of three separate assays. (B) Expressionof AQP1, GSH1 and MRPA analysed by real-time polymerase chain reaction (PCR)on RNA derived from K80SbIII and K135. Amplification was normalised to GAPDHprior to calculation of quantitative expression with respect to LdAG83, a standard

2 R. Singh et al. / International Journal

. donovani genomic microarray using a Maui mixer for 16 h at 42 ◦C.he hybridised microarrays were scanned in a laser scanner (Axon000A) and visualised using GenePixPro5.0 software. Replicatexperiments with three biological preparations were performedomparing antimony-resistant and -sensitive parasites. Data werenalysed with the help of Acuity 3.1 software following LOWESSormalisation.

.6. Semiquantitative reverse transcriptase (RT)-PCR

Semiquantitative RT-PCR was performed using RNA isolatedrom the same resistant and sensitive parasites. To check the reac-ion variability, pUC plasmid (1 pg/�L) was spiked in the cDNAreparation in equal ratio. Each reaction was amplified using gene-pecific primers (Table 1) as well as M13 forward and reverserimers (to amplify the 100-bp sequence from pUC plasmid). Con-rol experiments were performed with amplification using totalNA but no reverse transcriptase to rule out DNA contamination.

.7. Western blot analysis

Cell lysates (100 �g) from K80SbIII and K135 L. donovaniere subjected to sodium dodecyl sulphate polyacrylamide gel

lectrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and trans-erred to nitrocellulose membranes. The membrane strips werelocked, incubated sequentially with the primary antibodies andubsequently with anti-rabbit IgG or anti-mouse IgG conjugatedith HRP. Blots were developed using ECL reagent and were visu-

lised on X-ray film. The images were scanned and quantitativessessment was carried out with Image-J software (NIH IMAGE).

.8. Preparation of mutant parasites with episomal expression ofistone H2A in Leishmania

DNA encoding L. donovani H2A (LdH2A) obtained by ampli-ying L. donovani DNA was subcloned into pCR®2.1-TOPO TAloning vector. This recombinant DNA construct was termedCR®2.1-LdH2A. Sequence-confirmed plasmid DNA of pCR®2.1-OPO TA-fused LdH2A was used as template using respectiveene primers with SpeI site and HA tag for subcloning into theeishmania expression plasmid pKSNEO. The primer sequencesed for amplification of the full open reading frame of H2Aor cloning was forward, 5′-GGACTAGTATGGCTACTCCTCGCAG-GC-3′ and reverse, 5′-CCACTAGTCTACGCGTAGTCCGGCACGTCGTA-GGGTAAGCGCCCGGTGTCGCCTTGC-3′, with the SpeI site under-

ined and the HA tag in bold.The construct (20 �g) was transfected into one laboratory strain

LdS) and one field isolate (K133) by electroporation in 2-mm gapuvettes at 450 V and 500 mF to produce LdSH2A++ and K133H2A++,espectively. Transfectants were selected for resistance in G41850 �g/mL) as described previously [19]. Parasites transfected withhe empty vector pKSNEO were prepared as mock controls.

. Results

.1. Characterisation of Leishmania donovani clinical isolates

K80, adapted to grow at 125 �g/mL SbIII (K80SbIII), showedmedian effective dose (ED50) of >100 �g/mL SAG. K135 had an

D50 value of 4.22 ± 0.38 �g/mL [mean ± standard deviation (S.D.)]Fig. 1A).

Several mechanisms have been reported to contribute to anti-onial resistance in laboratory-generated resistant Leishmania

10]. Expression of the well-known antimony resistance media-or genes MRPA (encoding multidrug resistance protein A), GSH1encoding �-glutamylcysteine synthetase) and aquaglyceroporin

sensitive isolate. The graph shows the expression index defined as log ratios of geneexpression relative to that of LdAG83 (mean ± S.D. of three independent experi-ments).

(AQP1) (mediating drug uptake) was analysed by the quantitativereal-time PCR assay. All three genes were differentially expressedin strain K135 compared with K80SbIII, and the expression pat-tern was similar as reported earlier in the sensitive and resistantstrains, i.e. MRPA and GSH1 were overexpressed in the resistantstrain whilst AQP1 was preferentially expressed in the sensitiveparasite (Fig. 1B).

3.2. Expression profiling of drug-resistant genes

To understand the pattern of gene expression in SAG-sensitiveand -resistant L. donovani isolates, RNA isolated from K135 andK80SbIII Leishmania parasites, respectively, was used. A scatterplot of a representative hybridisation comparing expression inantimony-resistant and -sensitive parasites following LOWESS nor-malisation is shown in Fig. 2B. The scatter plot shows that mostspots are close to 0, indicating no change, but a fraction of thepoints deviate from the line of best fit, indicating possible differ-ential expression. Similar results were obtained in every (n = 4)experiment performed comparing resistant versus sensitive RNAexpression.

Analysis of microarray experiments revealed a number ofDNA clones showing differential expression in drug-resistant and-sensitive promastigotes. Robust statistical methods were thenapplied for these differentially expressed genes. Z-scores for eachgene were computed as the ratio of mean difference betweenthe two groups for each gene, divided by standard error for thecorresponding gene. Z-score values are used as the data basis incalculations of the Z-ratio. Of these, clones showing significant andconsistently higher expression with a ratio ≥1.4 in four microar-

ray hybridisations and reproducibility in dye flip microarraysexperiments were chosen. The higher the Z-score, the greater theconfidence that the transcript is differentially expressed betweenthe two phenotypes.

R. Singh et al. / International Journal of Antimicrobial Agents 36 (2010) 50–57 53

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ig. 2. Scatter plot showing the log ratios against average fluorescence intensities.arasites were hybridised to the microarray. Fluorescence intensities were measureverage of Cy3 and Cy5 intensities for each spot: (A) unnormalised data and (B) LOW

Clones meeting these criteria were selected and sequencedsing a single-pass automated sequencer for further analysis.urthermore, for their gene identity, the clone sequences wereearched by BLAST in the L. infantum genome. A total of 11 clonesut of 8448 were identified to be overexpressed in promastigotesf the resistant isolate K80SbIII and one gene was overexpressed inhe sensitive isolate K135 (Table 2). From the selected DNA clones,ve clones (68F10, 28F11, 90A5, 69E3 and 78B4) showed homologyith MAPK, two other clones (51E7 and 62D4) were homologous

o histone H4, one clone (87A9) was homologous to histone 2A,ne clone (47F10) showed homology to histone H1 and two clones58A8 and 87B9) showed homology to uncharacterised conservedypothetical protein-coding genes. From the clones downregulated

n resistant parasites, 42G8 showed homology to an amino acidransporter.

.3. Validation of microarray results by RT-PCR and Westernlotting

The differential expression observed in microarray analysis wasvident in RT-PCR analysis (Fig. 3). The fold difference in gene

able 2ifferentially expressed genes in resistant versus sensitive parasites as determined by mi

Clone ID Gene Gene DB systematic ID R/S

47F10 Histone H1 LinJ27 V3.1120 1.5387A9 Histone 2A LinJ29 V3.1860 1.7862D4 Histone H4 LinJ36 V3.0020 2.1451E7 Histone H4 LinJ36 V3.0020 1.7968F10 MAPK LinJ36 V3.6760 1.7028F11 MAPK LinJ36 V3.6760 1.6490A5 MAPK LinJ36 V3.6760 1.6269E3 MAPK LinJ36 V3.6760 1.5278B4 MAPK LinJ36 V3.6760 1.4242G8 Amino acid transporter LinJ31 V3.0350 0.6187B9 Hypothetical protein LinJ35 V3.3990, 73.1 kDa 1.4058A8 Hypothetical protein LinJ04 V3.0630, 23.3 kDa 1.70

, resistant; S, sensitive; S.D., standard deviation; MAPK, mitogen-activated protein kinas

abelled cDNA from drug-sensitive parasites and Cy5-labelled cDNA from resistanth a laser scanner and the log2 transformed intensity ratios were plotted against thenormalised data.

expression from the microarray as well as RT-PCR analysis is givenin Table 2 and the data are consistent in both the analyses.

The expression changes were further verified at the proteinlevel by Western blotting with the three available antibodies, H1,H2A and MAP kinase 1 (MAPK1). Expression at the protein levelmatched with expression at the RNA level, being 6-fold higher forH1, 2.5-fold for H2A and 5-fold for MAPK1 in resistant parasites incomparison with sensitive parasites (Fig. 4). Higher expression ofthese molecules at the transcript as well as protein level make themattractive candidates as drug resistance biomarkers to be tested infield isolates.

3.4. Overexpression of LdH2A in Leishmania

To see whether overexpression of one of the histone genes(LdH2A) would impart a drug-resistant phenotype in a drug-

sensitive parasite, LdH2A (which showed three-fold upregulationin resistant isolates by RT-PCR analysis) was overexpressed intwo SAG-susceptible L. donovani isolates, LdS and K133. LdH2Awas expressed with a HA tag. Overexpression was validated byWestern blotting of the total promastigote lysate probed with

croarray and reverse transcriptase polymerase chain reaction (RT-PCR) analysis.

median (± S.D.) ratio Z-ratio P-value RT-PCR fold change

± 0.22 3.55 0.001 1.52± 0.53 3.84 0.001 3.00± 0.50 3.14 0.001 2.50± 0.43 3.16 0.001 2.50± 0.42 3.92 0.001 3.50± 0.26 3.72 0.001 3.50± 0.45 2.16 0.003 3.50± 0.12 3.75 0.001 3.50± 0.22 2.44 0.002 3.50± 0.13 2.56 0.002 0.46± 0.26 1.49 0.01 2.00± 0.36 3.37 0.001 7.10

e.

54 R. Singh et al. / International Journal of Antimicrobial Agents 36 (2010) 50–57

Fig. 3. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of dif-ferentially expressed genes in antimony-sensitive K135 (S) and antimony-resistantK80SbIII (R) Leishmania parasites. In all the panels, the lower band (102 bp, pUC) rep-resents amplification of the pUC plasmid sequence spiked in the cDNA preparationfrom resistant or sensitive parasites, whilst the upper bands represent amplifica-tion of the gene sequence written below the panel. Negative control experimentswcs

enesiLtrociiy

Fig. 4. Validation of microarray results by Western blotting. The expression pat-tern was verified in the total promastigote lysate of the parasite used in themicroarray. Protein lysates (100 �g) from antimony-resistant K80SbIII (R) andantimony-sensitive K135 (S) were electrophoresed and probed with different pri-mary antibodies [histone H1, histone H2A and mitogen-activated protein kinase 1(MAPK1)] at a dilution of 1:500 and subsequently with secondary antibody rab-bit IgG conjugated with horseradish peroxidase (1:10 000). Blots were developedusing Western blot detection enhanced chemiluminescence (ECL) detection reagent.

FagcaHpc

ere performed with amplification using total RNA but no reverse transcriptase toheck for DNA contamination. Expression fold change in resistance with respect toensitive is shown in the term of x-fold.

ither anti-HA antibody (Fig. 5A) showing expression of the exoge-ous protein or anti-LdH2A antibody (Fig. 5B) showing both thendogenous and the exogenous proteins simultaneously. Expres-ion of H2A was 5–6-fold higher in LdSH2A++ and K133H2A++

n comparison with controls. Growth of the transfected parasitedSH2A++ and K133H2A++ was comparable with growth of con-rols transfected with the plasmid alone (LdSNeo and K133Neo,espectively), indicating that overexpression of H2A had no effectn promastigote viability (Fig. 5C). Assessment of infectivity by

ounting parasite-infected macrophages in 20 fields showed sim-lar (80%) infectivity in LdSH2A++ and K133H2A++ as well asn controls. Next, parasites overexpressing LdH2A were anal-sed for their susceptibility towards different antileishmanial

ig. 5. Characterisation of histone H2A-overexpressing Leishmania donovani isolates Ldnd K133H2A++ to confirm overexpression of H2A (16.2 kDa). Total promastigotes lysael electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membranes. The monjugated with horseradish peroxidase (HRP) and developed using Western blot detectntibody to monitor the amount of protein lysates loaded on the gel. (B) Western blot an2A compared with control. Total promastigotes lysates (100 �g) were separated on a 12robed with antibody to H2A followed by HRP-conjugated antibody and developed byontrols K133Neo and LdSNeo transfected with the plasmid alone. Each data point on the

Results shown are from a single experiment typical of at least three giving identicalresults. The blot was rebound with an �-tubulin antibody to monitor the amountof protein lysates loaded on the gel. Expression of (a) H1, (b) H2A and (c) MAPK1 inresistant (R) and sensitive isolates (S), respectively.

drugs as amastigotes. The ED50 (mean ± S.D.) of K133H2A++ andLdSH2A++ for SAG was 79.31 ± 2.47 �g/mL and 82.15 ± 7.12 �g/mL,respectively, which was significantly higher (P < 0.001) thantheir corresponding controls K133Neo (ED50 = 6.28 ± 0.92 �g/mL)and LdSNeo (ED50 = 6.37 ± 0.26 �g/mL) (Fig. 6A and D). Overex-pression of H2A decreased susceptibility of parasites not onlytowards SAG but also to AmB and MLF. The ED50 values ofK133H2A++ and LdSH2A++ for AmB were 0.89 ± 0.007 �g/mL and0.96 ± 0.01 �g/mL, respectively, which was significantly higher(P < 0.001) than the controls K133 Neo (0.24 ± 0.007 �g/mL) andLdS Neo (0.265 ± 0.007 �g/mL) (Fig. 6B,D). The ED50 for MLF wassignificantly higher (P < 0.05) for K133H2A++ (2.91 ± 0.26 �g/mL)and LdSH2A++ (2.66 ± 0.45 �g/mL) than their respective controls(1.4 ± 0.25 �g/mL and 1.46 ± 0.19 �g/mL) (Fig. 6C and D).

4. Discussion

SAG has been the first drug of choice against leishmaniasis.Resistance to this drug is a major problem in the field not only in the

SH2A++ and K133H2A++ (see Section 2.8). (A) Western blot analysis of LdSH2A++

tes (100 �g) were separated on a 12% sodium dodecyl sulphate polyacrylamideembrane was probed with anti-haemagglutinin antibody followed by rabbit IgGion enhanced chemiluminescence (ECL). The blot was rebound with an �-tubulinalysis for protein expression of histone H2A (16.2 kDa) in parasites overexpressing% SDS-PAGE gel and transferred to nitrocellulose membranes. The membrane was

using ECL. (C) Growth curve of K133H2A++ and LdSH2A++ in comparison with thecurve represents the mean ± standard deviation of three separate assays.

R. Singh et al. / International Journal of Antimicrobial Agents 36 (2010) 50–57 55

Fig. 6. Drug susceptibility of histone H2A-overexpressing Leishmania donovani isolates LdSH2A++ and K133H2A++ (see Section 2.8). Susceptibility of LdSH2A++ and K133H2A++

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as determined towards (A) sodium antimony gluconate (SAG), (B) amphotericin.1 macrophage cells. Cells transfected with plasmid alone (LdSNeo and K133Neo,

hree drugs for each strain of the parasites based on sigmoidal regression analysis beparate assays.

ndian subcontinent but also throughout the world [4,5,7–9]. Thencrease in resistance to SAG has led to an upsurge in therapeuticailures and, with limited chemotherapeutic alternatives, under-tanding the mechanisms responsible for resistance could help leado effective drug treatment strategies.

DNA microarrays have proved their utility in several studiesealing with the assessment of Leishmania gene expression dur-

ng parasite differentiation on a genome-wide scale that allowedhronicling of the molecular events during stage transition [18] asell as differences between KA and post kala-azar dermal leishma-iasis (PKDL) [20]. In the present study, DNA microarray technologyas employed to characterise alterations in gene expression that

ccur in drug-resistant and -sensitive L. donovani. The genomichotgun clones that were arrayed on the microarray used in thistudy represent an unbiased sample of the L. donovani genome.

Having established parasite resistance to the drug by observ-ng the relationship between clinical response and SAG sensitivityf K135 and K80 in vitro, transcriptome analysis on these isolatesas performed. Using rigorous statistical methods rather than sim-le fold changes for analysis of DNA microarray experiments, 11lones were identified that were upregulated in the resistant isolatend one clone (amino acid transporter) that was upregulated in theensitive isolate. BLAST analysis using L. infantum GeneDB revealedhat out of the 11 upregulated genes in the resistant isolate, fourelong to the histone family (H1, H2A and H4) and five were variousegments of the MAPK1 gene, whilst two of them were hypothet-cal proteins with unassigned functions. Furthermore, differentialxpression of these genes in sensitive and resistant parasites wasalidated by RT-PCR analysis. Expression patterns of three of these

enes were validated at the protein level.

Our observation of very few genes with modulated expres-ion in the resistant parasite is consistent with other studiesn drug-resistant parasites whereby whole-genome expressionrofiling revealed only 24 genes as differentially expressed in

B) and (C) miltefosine (MLF) as intracellular amastigotes in an assay using J774tively) were used as controls. (D) The median effective dose (ED50) for each of thein 6.0 software. Each data point represents the mean ± standard deviation of three

antimony-resistant parasites. In methotrexate-resistant parasites,again very few genes were differentially expressed without con-comitant change in copy number. In both the studies, modulation ingene expression was associated with gene amplification and genedeletion [16,21]. However, in resistant field isolates, researchersoften failed to detect gene amplification events, indicating thatalteration in RNA expression or point mutations may be responsiblefor the resistant phenotype [5,9,17].

MAPKs are well-known mediators of signal transduction ofhigher eukaryotes regulating important processes such as prolif-eration, differentiation, cell shape, stress response and apoptosis.Of the 17 MAPKs and MAPK-like kinases identified in Leishmania[22–24], we observed consistent upregulation of MAPK1 in theresistant isolate. MAPKs play a significant role in parasite intra-cellular proliferation, flagellar morphogenesis and hence parasitevirulence during mammalian infection [24–27]. Genetic studiesusing Leishmania mexicana MAPK null mutant parasites (LmxMPK)revealed that inactivation of LmxMPK1 abrogated parasite viru-lence during mammalian infection owing to a defect in intracellularproliferation, and null mutants of LmxMPK3 and LmxMPK9 showeddefects in flagellar morphogenesis that may have important conse-quences for sandfly infection and parasite transmission [24,26,27].We found five upregulated DNA clones mapping to MAPK1 in resis-tant isolates, emphasising its role in drug resistance. The higherexpression of MAPK1 was validated at transcript as well as proteinlevel. The exact mechanism of how MAPK1 contributes to antimonyresistance remains to be explored.

Leishmania parasites possess core histones H2A, H2B, H3,H4 [28,29] and linker histone H1 [30–32] that facilitate the

formation of higher-order chromatin structures. In eukaryotes,post-translational modifications of core histones act in diversebiological processes such as gene regulation, DNA repair and chro-mosome condensation (mitosis). Histone synthesis in Leishmaniais tightly coupled to DNA replication by a post-transcriptional

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6 R. Singh et al. / International Journal

echanism operating at the level of translation [33]. Very fewtudies have been carried out on trypanosomatid histone modi-cations and on their role in gene regulation. Recently, a ChIP–chipssay revealed that H3 histones at the origins of polycistronicranscription of protein-coding genes are acetylated and possibly

odification in the acetylation state of these origins regulates tran-cription initiation [34]. However, the role of histone proteins inntimony resistance has not been established. In the present study,f the 11 DNA clones upregulated in resistant parasites, four cor-esponded to histones, namely H1, H2A and H4. In Leishmania,istone mRNA levels are regulated by a mechanism coupled toellular growth. During promastigote growth, histone H1 mRNArogressively accumulates from early log phase to stationary phase.ddition of histone H1 affects chromatin condensation of para-ite nuclei and its overexpression in Leishmania has resulted in theeduced infectivity of the parasite in vitro as well as in vivo [35,36].

e observed 1.5-fold upregulation of histone H1 at the RNA level inresistant isolate and more than 6-fold upregulation at the protein

evel.Up to 2-fold changes in expression of core histones H2A and H4

as observed by microarray, and similar results were obtained byT-PCR. Expression of H2A was found to be elevated at the protein

evel, signifying its functional role. Unlike histone H1, a decreasen RNA levels associated with the growth phase has been observedor other L. infantum histones H2A, H3 and H4 [28,29,37]. Identicalrowth points were used for comparative expression profiling elim-nating the possibility of growth-related differential expression ofistones.

Transfection studies using overexpression of a gene have provedheir utility in several studies dealing with assessment of Leish-ania phenotypes. Overexpression of HSP83 and P299 gene in the

ensitive L. donovani isolate conferred increased resistance to triva-ent antimony and MLF [15,38]. Here we showed that transfectionf histone H2A in two sensitive isolates of L. donovani originat-ng from distinct geographic regions conferred a more than 12-foldncrease in SAG resistance. In addition, it also conferred resistanceo other antileishmanial drugs such as AmB and MLF, albeit slightlyower than SAG (ca. 4-fold and 2-fold, respectively) in comparison

ith the wild-type. Finally, our results suggest that overexpressionf histone H2A, if observed in field isolates, will indicate not onlyesistance to antimonials but also lower susceptibility to AmB andLF, especially in India where plausible cross-resistance appears

o operate in field isolates [6].Demonstration that higher expression of the nucleosome core

istone H2A is found in clinically isolated SAG-resistant parasitesnd that forced overexpression of this protein in susceptible strainsonverts them to resistant suggests a mechanism of resistance notreviously recognised. The well-established role of histone H2A inNA packaging opens up a new area of investigation to overcomerug resistance and to develop more effective antileishmanials.oreover, the expression analysis of identified genes in field iso-

ates may serve as potential biomarkers of drug resistance, useful touide therapy and prevent the use of possibly ineffective and toxicgents in VL patients.

cknowledgments

Critical suggestions by G. Sreenivas (CBER, FDA, Bethesda, MD)re gratefully acknowledged. The authors are thankful to Jose. Requena (Universidad Autónoma de Madrid, Spain) for anti-2A antibodies, to Ketty P. Soteriadou (Hellenic Pasteur Institute,

thens, Greece) for anti-H1 antibodies and to Martin Wiese (Bern-ard Nocht Institute for Tropical Medicine, Hamburg, Germany) fornti-MAPK1 antibodies.

Funding: This work was supported by an Indian Council of Med-cal Research (ICMR, India) grant to PS and a UNESCO L’Oreal for

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imicrobial Agents 36 (2010) 50–57

Women in Science fellowship grant to RS. DK is grateful to theCouncil of Scientific and Industrial Research (CSIR) for financialsupport.

Competing interests: None declared.Ethical approval: This work was carried out under ethical

approval and guidelines of the Ethical Committee of SafdarjungHospital (New Delhi, India).

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