ORIGINAL PAPER Comparative transcript expression analysis of miltefosine-sensitive and miltefosine-resistant Leishmania donovani Arpita Kulshrestha & Vanila Sharma & Ruchi Singh & Poonam Salotra Received: 17 October 2013 /Accepted: 3 January 2014 /Published online: 22 January 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Leishmania donovani is the causative agent of anthroponotic visceral leishmaniasis in the Indian subconti- nent. Oral miltefosine therapy has recently replaced antimo- nials in endemic areas. However, the drug is at risk of emer- gence of resistance due to unrestricted use, and, already, there are indications towards decline in treatment efficacy. Hence, understanding the mechanism of miltefosine resistance in the parasite is crucial. We employed genomic microarray analysis to compare the gene expression patterns of miltefosine- resistant and miltefosine-sensitive L. donovani . Three hundred eleven genes, representing ∼3.9 % of the total Leishmania genome, belonging to various functional categories including metabolic pathways, transporters, and cellular components, were differentially expressed in miltefosine-resistant parasite. Results in the present study highlighted the probable mecha- nisms by which the parasite sustains miltefosine pressure including (1) compromised DNA replication/repair mecha- nism, (2) reduced protein synthesis and degradation, (3) al- tered energy utilization via increased lipid degradation, (4) increased ABC 1-mediated drug efflux, and (5) increased antioxidant defense mechanism via elevated trypanothione metabolism. The study provided the comprehensive insight into the underlying mechanism of miltefosine resistance in L. donovani that may be useful to design strategies to increase lifespan of this important oral antileishmanial drug. Introduction Leishmania donovani is the etiological agent of visceral leish- maniasis (VL), a potentially fatal systemic protozoal infection. The emergence and spread of resistance to therapy for VL is of significance particularly in India where more than 60 % of patients do not respond to the traditional antimonial therapy. Miltefosine (hexadecylphosphocholine) is an oral drug initially developed as an anticancer agent for the treatment of cutaneous lymphomas and breast cancer that shows selec- tive activity against Leishmania (Clive et al. 1999; Croft et al. 2006). In India, miltefosine has recently taken over as the first- line therapy for VL even in areas where antimonials are effective (WHO TDR News 2004). However, widespread use of miltefosine monotherapy might lead to the rapid emer- gence of resistance in India, where VL is anthroponotic (Bryceson 2001). The long half-life of the drug may poten- tially increase the risk of development of experimental resis- tance to this drug shown to be readily induced in vitro (Seifert et al. 2003). Concerns have been raised over rise in miltefosine treatment failure and relapses (almost double) in phase IV clinical trials in India (Sundar et al. 1998; Sundar and Murray 2005). Already, reports of clinical failure and relapse have come up in India and Nepal (Sundar et al. 2006; Pandey et al. 2009). In this situation, it becomes important to under- stand the mechanism of action and development of resistance towards miltefosine in the parasite. The mechanisms and related biological pathways that con- tribute to miltefosine resistance in the parasite are relatively poorly understood. Suggested targets of miltefosine in Leishmania include perturbation of ether-lipid metabolism, glycosyl phosphatidylinositol anchor biosynthesis, signal Electronic supplementary material The online version of this article (doi:10.1007/s00436-014-3755-6) contains supplementary material, which is available to authorized users. A. Kulshrestha : V. Sharma : R. Singh : P. Salotra (*) National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, India e-mail: [email protected]Present Address: A. Kulshrestha Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Parasitol Res (2014) 113:1171–1184 DOI 10.1007/s00436-014-3755-6
Transcript
ORIGINAL PAPER
Comparative transcript expression analysis of miltefosine-sensitiveand miltefosine-resistant Leishmania donovani
Received: 17 October 2013 /Accepted: 3 January 2014 /Published online: 22 January 2014# Springer-Verlag Berlin Heidelberg 2014
Abstract Leishmania donovani is the causative agent ofanthroponotic visceral leishmaniasis in the Indian subconti-nent. Oral miltefosine therapy has recently replaced antimo-nials in endemic areas. However, the drug is at risk of emer-gence of resistance due to unrestricted use, and, already, thereare indications towards decline in treatment efficacy. Hence,understanding the mechanism of miltefosine resistance in theparasite is crucial. We employed genomic microarray analysisto compare the gene expression patterns of miltefosine-resistant and miltefosine-sensitive L. donovani. Three hundredeleven genes, representing ∼3.9 % of the total Leishmaniagenome, belonging to various functional categories includingmetabolic pathways, transporters, and cellular components,were differentially expressed in miltefosine-resistant parasite.Results in the present study highlighted the probable mecha-nisms by which the parasite sustains miltefosine pressureincluding (1) compromised DNA replication/repair mecha-nism, (2) reduced protein synthesis and degradation, (3) al-tered energy utilization via increased lipid degradation, (4)increased ABC 1-mediated drug efflux, and (5) increasedantioxidant defense mechanism via elevated trypanothionemetabolism. The study provided the comprehensive insightinto the underlying mechanism of miltefosine resistance in
L. donovani that may be useful to design strategies to increaselifespan of this important oral antileishmanial drug.
Introduction
Leishmania donovani is the etiological agent of visceral leish-maniasis (VL), a potentially fatal systemic protozoal infection.The emergence and spread of resistance to therapy for VL is ofsignificance particularly in India where more than 60 % ofpatients do not respond to the traditional antimonial therapy.
Miltefosine (hexadecylphosphocholine) is an oral druginitially developed as an anticancer agent for the treatmentof cutaneous lymphomas and breast cancer that shows selec-tive activity against Leishmania (Clive et al. 1999; Croft et al.2006). In India, miltefosine has recently taken over as the first-line therapy for VL even in areas where antimonials areeffective (WHO TDR News 2004). However, widespreaduse of miltefosine monotherapy might lead to the rapid emer-gence of resistance in India, where VL is anthroponotic(Bryceson 2001). The long half-life of the drug may poten-tially increase the risk of development of experimental resis-tance to this drug shown to be readily induced in vitro (Seifertet al. 2003). Concerns have been raised over rise in miltefosinetreatment failure and relapses (almost double) in phase IVclinical trials in India (Sundar et al. 1998; Sundar andMurray 2005). Already, reports of clinical failure and relapsehave come up in India and Nepal (Sundar et al. 2006; Pandeyet al. 2009). In this situation, it becomes important to under-stand the mechanism of action and development of resistancetowards miltefosine in the parasite.
The mechanisms and related biological pathways that con-tribute to miltefosine resistance in the parasite are relativelypoorly understood. Suggested targets of miltefosine inLeishmania include perturbation of ether-lipid metabolism,glycosyl phosphatidylinositol anchor biosynthesis, signal
Electronic supplementary material The online version of this article(doi:10.1007/s00436-014-3755-6) contains supplementary material,which is available to authorized users.
A. Kulshrestha :V. Sharma : R. Singh : P. Salotra (*)National Institute of Pathology (ICMR), Safdarjung HospitalCampus, New Delhi 110029, Indiae-mail: [email protected]
Present Address:A. KulshresthaRosalind Franklin University of Medicine and Science,North Chicago, IL, USA
Parasitol Res (2014) 113:1171–1184DOI 10.1007/s00436-014-3755-6
transduction as well as inhibition of acyl transferase, an en-zyme involved in lipid remodeling (Lux et al. 2000). Currentevidence suggests that the drug kills Leishmania cells by aprocess reminiscent of programmed cell death (Verma andDey 2004; do Monte-Neto et al. 2011). An impairment indrug uptake machinery involving amino-phospholipidtranslocase miltefosine transporter (LdMT) and an accessoryprotein, LdRos3 (CDC50/Lem3 family) in experimentalmiltefosine-resistant Leishmania lines was proposed to bethe most likely mechanism of resistance (Perez-Victoriaet al. 2006). Proteomic analysis revealed role of eukaryoticinitiation factor eIF4 in miltefosine resistance in L. donovani(Singh et al. 2008). Regardless, a better understanding of themolecular mechanism involved is of paramount importance.
The completion of the genomic sequences of severalLeishmania species (http://www.genedb.org/) provided theopportunity to study the pattern of whole-genome differentialexpression during drug resistance. In recent times, gene ex-pression microarray has become a well-established technolo-gy by which the expression of thousands of genes can bemeasured simultaneously providing a global genetic perspec-tive on complex biological processes like drug resistance.Various studies have demonstrated the usefulness of whole-genome DNA microarrays for studying drug resistance inLeishmania (Ubeda et al. 2008; Leprohon et al. 2009; Singhet al. 2010). However, the modulations in Leishmania tran-scriptome in miltefosine resistance are poorly explored.
The current study utilized whole-genome Leishmania spp.oligonucleotide array to explore differences in gene expres-sion between miltefosine resistant and sensitive L. donovaniparasite. In this study, populations of L. donovani resistant tomiltefosine were selected in vitro in order to study global geneexpression modulation associated with resistance. In-depthbioinformatic analysis was performed to identify changes ingroups of interacting genes or pathways that may contribute toresistance to miltefosine. We have found evidence of alteredexpression of several genes belonging to DNA repair andreplication machinery, protein translation and folding, energygeneration by lipid degradation, transporter activity, and anti-oxidant defense mechanism in miltefosine-resistantL. donovani parasite.
Materials and methods
Parasite culture
Parasite isolates were prepared from bone marrow aspirate ofVL patient originating from Bihar and reporting to SafdarjungHospital (SJH), New Delhi, as described previously(Kulshrestha et al. 2011). Informed consent was obtained frompatients according to the guidelines of the Ethical Committee,SJH. This wild-type parental line (miltefosine-sensitive cell
lines referred as MIL-S1 MIL-S2 and MIL-S3) were culturedas promastigotes in Medium 199 (Sigma Aldrich, USA),25 mM HEPES N-[2-hydroxyethyl]piperazine-N−1-[2-ethanesulfonic acid] (Sigma Aldrich, USA), 100 IU, and100 μg/ml each of penicillin G (Sigma Aldrich, USA) andstreptomycin sulphate (Sigma Aldrich, USA), respectively,supplemented with 10 % heat- inactivated fetal calf serum(FCS; Gibco, USA) at 26 °C and 7.4 pH. The parasites werecharacterized as L. donovani by species-specific PCR (Salotraet al. 2001).
Preparation of miltefosine stock/drug stock
Miltefosine (Cayman Chemical Company, USA) stock wasprepared by dissolving the drug at concentration of 5 mg/ml inabsolute methanol and stored at 4 °C up to 1 month. Theworking stock was prepared fresh in Medium 199 on the dayof experiment.
Generation and characterization of experimental L. donovanistrains resistant to miltefosine
Wild-type parental L. donovani lines (MIL-S1, MIL-S2, andMIL-S3) were adapted to grow under high MIL pressure byin vitro passage with a stepwise increase in the miltefosineconcentration (2.5, 5, 7.5,10, 20, and 30 μg/ml) in mediumM199 to generate miltefosine-resistant parasite designated asMIL-R1, MIL-R2, and MIL-R3. At each step, parasites werecultured for at least five to eight passages to attain steady andoptimal cell growth comparable to its wild-type miltefosine-sensitive counterpart. TheMIL-R1 line was taken up further formicroarray analysis, while validation of microarray data byreal-time PCR was done in three miltefosine-resistant line(MIL-R1, R2, and R3). The susceptibility of miltefosine-resistant L. donovani (MIL-R1) to current antileishmanial drugs(miltefosine, SAG, amphotericin B, paromomycin, andsitamaquine) was tested at intracellular amastigote stage asdescribed previously (Singh et al. 2010; Kulshrestha et al.2011). Single-nucleotide polymorphism, in LdMT andLdRos3 genes, associated with miltefosine resistance was de-termined for both sensitive and resistant cell lines. The geneswere PCR amplified and amplicons were sequenced onAutomated Sequencer ABI 3730. Primers used for amplifica-tion and sequencing are given as ESM Tables S1.1 and S1.2.
Oligonucleotide array
One-color microarray-based gene expression profiling was car-ried out using a high-density Leishmania multispecies 60-meroligonucleotide microarray slide [8×15K format] representingthe entire genome of Leishmania infantum and Leishmaniamajor. The microarray chip, printed by Agilent Technologies,USA, included a total of 9,233 Leishmania-specific genes
including 540 control probes. Gene expression analysisemploying this array has been described previously (Ubedaet al. 2008; Leprohon et al. 2009; Rochette et al. 2008).
RNA isolation
Total RNA was extracted from 108 late log phasepromastigotes using Trizol reagent according to manufac-turer’s instructions. RNA clean up was performed usingRNeasy Plus mini kit (Qiagen, Gaithersburg, MD, USA) asdescribed by the manufacturer. The purified RNAwas quan-tified using Nanodrop by estimating the absorbance at 260 and280 nm. The quality and integrity of RNA was assessed onRNA 6000 Nano Assay Chips on Bioanalyzer 2100 (AgilentTechnologies, Santa Clara, CA, USA). The presence of threedistinct ribosomal peaks (18S, 24Sα, and 24Sβ) confirmedsuccessful RNA extraction.
RNA labelling and microarray hybridization
Complementary RNA (cRNA) was generated from 1 μg oftotal RNA using Quick-Amp Labeling kit (AgilentTechnologies) that directly incorporates Cy-3 labeled CTPinto the cRNA. Prehybridization and hybridization were per-formed according to manufacturers’ instructions. Labeledprobes were hybridized with Leishmania oligonucleotide ar-ray using Gene Expression Hybridization Kit (AgilentTechnologies) at 65 °C for 17 h. The hybridized arrays weresubsequently washed with gene expression hybridizationbuffers 1 and 2 using 0.005 % Triton X-102. Experimentswere performed using three independent RNA extractions.The slides were scanned immediately in Axon GenePix4000B scanner to minimize the impact of environmentaloxidants on signal intensities. GeneSpring GX 11.0.2 micro-array data and pathway analysis tool was used for data anal-ysis. Analysis involved data preprocessing; elimination ofoutliers, nonsignificantly expressed genes and false positives;and analysis of the gene lists in a biological perspective. Thedata files were in text (.txt) format and obtained fromAgilent’sFeature Extraction (FE). The summarization of the “raw”signal values was performed by computing the geometricmean. “Normalized” value was generated after log transfor-mation and normalization (scale) and baseline transformation.For each probe, the median of the log summarized values fromall the samples is calculated and subtracted from each of thesamples. Quartile (75th percentile) normalization was per-formed. Storey and bootstrapping analysis was performedfor multiple testing corrections. Statistically significant differ-entially expressed genes were determined by t test (unpaired)for two groups; (p value cutoff)<0.05. All microarray data isavailable on the GEO NCBI database in the MIAME format;http://www.ncbi.nlm.nih.gov/geo/ with the GEO accessionnumber GSE 303685. Genes with expression ratio greater
than 2.0 between wild-type and miltefosine-resistant parasitewere considered as differentially expressed were clusteredusing hierarchical clustering based on Pearson coefficientcorrelation algorithm. DNA microarray data were analyzedby custom R programs to illustrate the expression profile ofMIL-R L. donovaniby extrapolating on a chromosomemap ofL. infantum. Gene ontology annotation for functional classifi-cation of modulated genes was performed using GeneDB,BLAST2GO, and AmiGO databases. The pathway analysiswas carried out using GeneSpring GX11.0 and KEGG path-way analysis tool. String 9.05 database (string-db.org) wasemployed to understand the interaction network/patterns ofdifferentially regulated genes at protein level.
Quantitative RT-PCR
Q-RT-PCR was carried out on a selected number of genes(ESM Table S2) in order to validate the microarray experi-ments in three MIL-R lines (MIL-R1, R2, and MILR 3)generated from different parental lines to confirm the associ-ation of these genes with miltefosine resistance. Briefly, first-strand cDNA was synthesized from 5 μg of total RNA ofMIL-R/MIL-S parasites using the Superscript II RNAse Hreverse transcriptase enzyme (Invitrogen, Carlsbad, CA,USA) and Oligo dT primers (Fermentas, USA) according tothe manufacturer’s protocol. Three independent RNA prepa-rations were used for each Q-PCR experiment. Equal amountsof cDNAwere run in triplicate and amplified in 20μl reactionscontaining 1× Fast SYBR Green Mastermix (AppliedBiosystems, USA), 100 ng/ml forward and reverse primers.Reactions were carried out using ABI 7500 Real-Time PCRsystem (Applied Biosystems, USA). Initially, mixtures wereincubated at 95 °C for 10 min and then cycled 40 times at95 °C for 15 s and 60 °C for 1 min. No-template controls wereused as recommended. The relative amount of PCR productsgenerated from each primer set was determined based on thethreshold cycle (Ct) value and the amplification efficiencies.Gene expression levels were normalized to constitutivelyexpressed mRNA encoding glyceraldehyde-3-phosphate de-hydrogenase (GAPDH). Quantification of the relative changesin target gene expression was calculated using −2ΔΔCt method.Primers for targeted genes were designed using Primer ex-press software version 3.0 (Applied Biosystems, USA) andoligo-analyzer (Integrated DNATechnologies, USA).
Results
Characterization of laboratory-generated miltefosine-resistantL. donovani parasites
L. donovani field isolates were selected for miltefosine resis-tance up to 30 μg/ml drug pressure. The ED50±SEM value of
the MIL-S1 at promastigote stage was 7.02±0.18 μg/ml;whereas the miltefosine-resistant mutant MIL-R1 exhibited aninefold higher ED50 of 64.38±2.17 μg/ml (Table 1) com-pared to the corresponding wild-type isolate. The miltefosineresistance was stable even in the absence of drug pressure forover 12 passages. The resistance induced at promastigotestage was also evident at amastigote stage using J774.A.1murine macrophage cell line. No significant change was ob-served in the susceptibility of MIL-R parasite towards otherantileishmanial drugs (Table 1). The susceptibility ofpromastigote and amastigote forms towards miltefosine wasin good correlation (Seifert et al. 2003; Kumar et al. 2009),hence the promastigote stage of the parasite was established asan efficient model for this study.
Sequence analysis of LdMT genes revealed the previouslyreported single-nucleotide polymorphism, C1259→Aresulting in substitution of Thr 420→Asn in the MIL-R celllines. Additionally, a novel SNP, T 527→A resulting insubstitution of Val 176→Asp and in LdMT gene of resistantcell lines was observed. A nucleotide exchange leading toconversion of valine to aspartic acid is significant toperturbing protein’s structural stability. However, no pointmutations were detected in case of LdRos.
mRNA expression profiling of miltefosine-resistantL. donovani promastigote
The whole-genome Leishmaniamicroarray analysis revealed atotal of 311 differentially expressed genes representing 3.9% ofthe total Leishmaniagenome inMIL-R parasite (taking twofoldcutoff). Normalized intensity scatter plot in a base 2 logarithmicscale is shown in Fig. 1. Out of 311 genes, 184 genes (∼2.2 %)were preferentially expressed in MIL-R L. donovani, 127 genes(∼1.72 %) were found to be preferentially expressed in MIL-Sparasite. Majority of these genes showed an average change inmRNA accumulation not exceeding threefold (Table 2).
The data generated from the MIL-R Leishmania microarrayexperiments is illustrated by a chromosome map representinggene expression levels on a genomic scale (Fig. 2). A closeanalysis of the normalized expression data generated by themicroarray experiments confirmed that most genes located onthese chromosomes were modulated by a factor ranging from 2to 2.5 in mRNA abundance. The expression of genes on allchromosomes, except chromosomes 1, 2, and 3, were modulatedmore than twofold in MIL-R L. donovani, as suggested by thechromosomemap of gene expression. All modulated genes fromchromosomes 5, 15, 23, 26, and 29 were upregulated (represent-ed in red) while those of chromosome 6 were downregulated(represented in green). Interestingly, it was found that genesupregulated in miltefosine-induced resistance were mainly con-fined to chromosome numbers 15 and 23 as illustrated in Fig. 2.Table 1 Susceptibility of miltefosine resistant L. donovani mutant to-
SAG sodium antimony gluconate, ND not determinedaPro Promastigotes; values represent mean of ED50±SEM of two inde-pendent experiments performed in triplicatebAmasAmastigotes; values represent mean of ED50±SEM of two inde-pendent experiments performed in triplicate at intracellular amastigotestage in J774A.1 cell linec The drug sensitivity could not be determined above this concentration asmiltefosine poses cytotoxic effect on the host macrophages above theconcentration of 30 μg/ml
*p<0.05, ED50 considered significantly different
Fig. 1 Normalized scatter plot of microarray gene expression analysisdata. The scatter plot represents normalized average signal intensityvalues (log scale) comparing MIL-R (Y-axis) versus wild-type control(X-axis). The position of each dot on the scatter plot corresponds to thenormalized average signal intensity of a single gene. Each point repre-sents an individual gene that is either upregulated in MIL resistant-relative to MIL sensitive (upper left quadrant) or that is downregulatedin MIL-resistant relative to MIL sensitive (lower right quadrant). Thecenter line represents no difference in expression level for a given genebetween the MIL sensitive and MIL resistant while the upper and lowerline indicate the 2 fold change cut off used. 3.9 % of genes were outsidethese fold-change thresholds. Linear regression analysis was performed
1174 Parasitol Res (2014) 113:1171–1184
The chromosomal map representation enabled the identifi-cation of a locus on chromosome 23 that was over expressed inMIL-R parasite (Fig. 2). The upregulated genomic region com-prised of the genes hydrophilic acylated surface protein A(HASPA1 and A2) and small hydrophilic endoplasmicreticulum-associated protein (SHERP) which were all substan-tially upregulated in MIL-R parasite. SHERP proteins are re-ported to be metacyclic stage-specific and involved in mem-brane trafficking. Among the genes upregulated on chromo-some 29, Tob55, a member of topogenesis of mitochondrialouter membrane β-barrel proteins (TOB complex, also called
the sorting and assembly machinery) (Paschen et al. 2003;Gentle et al. 2004) was found upregulated in MIL-R parasitepossibly modulating translocation of ions and small moleculesacross the mitochondrial membrane in the resistant parasites.Significant genes upregulated on Chr5-included motor proteins(dynein light chain and kinesin-like protein), ATPase, andsurface antigen-like protein. The ABCA7 transporters, proteinkinase C, and tryperidoxin peroxidase were among the majorgenes preferentially expressed on chromosome 15 in MIL-Rparasite. All 17 genes upregulated on Chr26 were hypothetical.
Pathway analysis
BLAST2GO, AmiGO, and KEGG pathway analysis indicatedupregulation of DNA synthesis and transporter activities anddownregulation of protein metabolism in the MIL-R parasite.The percentage of differentially modulated genes according togene ontology (GO) function categories and variousmetabolicprocesses is shown in Fig. 3. The changes in expression levelof several genes belonging to various categories describedbelow are listed in Table 3. The interaction among the genesat protein level was analyzed using STRING 9.05 database.
DNA replication and repair mechanism
The expression of genes involved in DNA replication andrepair mechanism was found to be significantly affected.
Table 2 Patterns of Global mRNA expression in miltefosine resistantL. donovani parasite
mRNA upregulation(fold increase)
MIL-resistantL. donovani
Miltefosine-sensitiveL. donovani
2.0–2.5 90 89
2.5–3.0 47 19
3.0–3.5 23 10
3.5–4.5 16 9
4.5–6.0 8 –
Total genes 184 127
Percentage of modulated genes 2.3 % 1.6 %
The percent modulated genes calculated from the total 7,963 genesobtained in QC after filtering
Fig. 2 Chromosome wise modulation of gene expression in miltefosineresistant L. donovani. DNA microarray data were analyzed by custom Rprograms to illustrate the expression profile of L.donovaniMIL-R/WT byextrapolating on a chromosome map of L. infantum. Red lines indicateupregulated genes (greater than twofold upregulated) in MIL-R parasite,whereas green lines indicate downregulated genes (less than twofolddownregulated), gray lines are the genes exhibiting less than twofold
expression in both conditions. Chromosomes showing high upregulationinmiltefosine resistance included Chr 5, 15, and 23. The area encircledonchromosome 23 represents the locus containing genes SHERP andHASPA1. Likewise encircled locuson Chr 15 represents genes of nucleicacid synthesis and repair mechanism; on Chr 5 genes coding for cyto-skeleton molecules are encircled
Parasitol Res (2014) 113:1171–1184 1175
The expression of DNA replication licensing factor(LinJ28.2490), Endo/exonuclease Mre11 (LinJ27.1550), andA/G-specific adenine glycosylase (LinJ28.2230) was down-regulated in MIL-R parasites. On the contrary, proliferativecell nuclear antigen (PCNA- LinJ15.1410) and RAD51/dmc1protein (LinJ35.5000) were overexpressed in miltefosineresistance.
Protein synthesis, folding, and secretion
MIL-S or wild-type parasite exhibited an activated proteinsynthesis in comparison toMIL-R parasite. Chaperone proteinDnaJ and ubiquitin-activating enzyme E1 involved in proteo-lytic process were downregulated in miltefosine resistance.Chaperonin TCP20 (LinJ13_V3.1400), a protein involved infolding process of proteins was also downregulated inmiltefosine resistance. Reduced expression of isoleucyl-tRNA synthetase transcript involved in aminoacyl-tRNA bio-synthesis during translation indicated downregulated tRNAbiosynthesis in MIL-R parasite. Protein-degrading enzymeswere also reduced in miltefosine resistance as suggested bydownregulated expression of proteolytic metallopeptidasesencoding genes including carboxypeptidase, metallo-peptidase, and thimet oligopeptidase. Cathepsin L-like prote-ases (LinJ08_V3.0960) were, however, upregulated in MIL-Rparasite. Among the genes involved in protein processing inER, a hypothetical protein and a putative transitional ERATPase were downregulated in miltefosine resistance.
Carbohydrate metabolism and oxidative phosphorylation
Beta-fructosidase-like protein, invertase-like protein, sucrosehydrolase-like protein (LinJ23_V3.1060) were upregulated inMIL-R parasite while 2-hydroxy-3-oxopropionate reductase,(LinJ30_V3.0170) was downregulated. Enzyme phospho-glycerate mutase (LinJ08.0060) and Acetyl CoA synthase(LinJ23.0580) were found to be upregulated in MIL-R para-site, while Pyruvate dehydrogenase E1 beta subunit(LinJ25.1790) was downregulated. Genes involved in oxida-tive phosphorylation including ATP synthase (LinJ36.3790)and vacuolar ATP synthase subunit B (LinJ28.2540) weredownregulated in MIL-R parasite.
Fatty acid biosynthesis and lipid degradation
Two genes associated with lipid metabolic process includingl ipase (L inJ31_V3.2540) and l ipase precur sor(LinJ31_V3.0870) like protein were upregulated, while fattyacid elongase (LinJ14_V3.0700), NADH-cytochrome B5 re-ductase (LinJ22_V3.0590), and 2,4-dienoyl-CoA reductase-like protein (LinJ06_V3.0960) were downregulated inmiltefosine resistance.
Transport and drug resistance
Interestingly, several genes involved in transport andtransporter-like activity were upregulated in MIL-R parasite
Fig. 3 Distribution of genes differentially modulated during miltefosineresistance in Leishmania donovani according to gene ontology (GO)function categories. a Overall distribution of GO categories of genesdifferentially expressed in MIL-R parasite suggest that genes belongingto metabolic process, transport, cell component, and organization were
affected. bDifferent metabolic processes that were modulated in MIL-Rparasite include DNA, nucleotide, nucleobase, protein and lipids.Unclassified proteins include the hypothetical proteins (proteins withunknown function and not tested experimentally) and proteins with noGO category (unclassified) that have been experimentally characterized
Nucleic acid metabolic process [GO:0006259, GO:0006139, GO:0032774]
1 LinJ15_V3.1500 2.50 Up Proliferative cell nuclear antigen (PCNA), putative
2 LinJ29_V3.2710 3.22 Up Poly(A) polymerase, putative
3 LinJ23_V3.1210 3.85 Up Small hydrophilic endoplasmic reticulum-associated protein (SHERP)
4 LinJ35_V3.4950 2.09 Up RAD51/dmc1 protein (DMC1)
5 LinJ19_V3.0470 2.58 Up Nuclear cap binding complex subunit CBP30
6 LinJ21_V3.0690 2.38 Down DNA polymerase eta, putative
7 LinJ27_V3.1790 2.23 Down Endo/exonuclease Mre11, putative
8 LinJ28_V3.2550 2.93 Down DNA replication licensing factor, putative
9 LinJ11_V3.0540 2.07 Down Hypothetical protein, conserved, pseudouridine synthesis
10 LinJ17_V3.0930 2.14 Down Hypothetical protein, conserved
11 LinJ21_V3.0600 2.48 Down la RNA binding protein, putative
12 LinJ22_V3.1110 2.17 Down Ribonucleoside-diphosphate reductase small chain, putative
13 LinJ25_V3.1210 2.48 Down ATPase beta subunit, putative
14 LinJ28_V3.2290 2.56 Down A/G-specific adenine glycosylase, putative
15 LinJ28_V3.2450 2.22 Down DNA topoisomerase ii
16 LinJ32_V3.3930 2.80 Down Kinetoplast DNA-associated protein, putative
Proteinmetabolic process and catalytic activity [GO:0019538, GO:0006464, GO:0006508, GO:0003824]Protein metabolic process and catalytic activity[GO:0019538]
17 LinJ08_V3.0960 2.19 Up Cathepsin L-like protease
18 LmjF15.1480 2.57 Up cAMP specific phosphodiesterase, putative
19 LinJ23_V3.0580 2.08 Up Acetyl-CoA synthetase, putative
20 LinJ23_V3.0080 2.57 Up Agmatinase-like protein
21 LinJ20_V3.1740 2.49 Up Aminoacylase, putative, N-acyl-L-amino acid amidohydrolase, putative
22 LinJ25_V3.1540 2.80 Up Calpain family cysteine protease-like protein
23 LinJ23_V3.0700 2.16 Up Hypothetical protein
24 LmjF23.1665 2.00 Up Hypothetical protein
25 LinJ29_V3.2880 2.73 Up Hypothetical protein, conserved
26 LinJ15_V3.0050 3.53 Up Cytochrome-b5 reductase, putative
27 LinJ09_V3.1120 2.16 Up DNA-directed RNA polymerase III subunit, putative
28 LinJ29_V3.1670 2.25 Up GTPase activator protein, putative
29 LinJ19_V3.1670 2.86 Up Hypothetical protein, unknown function
30 LinJ15_V3.0170 2.80 Up Protein phosphatase 2C, putative
31 LinJ26_V3.1800 2.02 Up Hypothetical protein, conserved
32 LinJ10_V3.1360 3.22 Up Hypothetical protein
33 LinJ08_V3.0060 2.82 Up Phosphoglycerate mutase protein, putative
34 LinJ15_V3.1630 2.87 Up Protein kinase, putative
35 LinJ05_V3.1210 2.02 Up Surface antigen-like protein
36 LinJ22_V3.0630 3.08 Up Protein kinase putative, serine/threonine protein kinase sos2, putative
37 LinJ08_V3.0870 4.42 Up Protein kinase, putative
38 LinJ23_V3.0500 2.45 Up Trypanothione synthetase, putative
39 LinJ15_V3.1540 3.06 Up cAMP specific phosphodiesterase
40 LinJ15_V3.1120 2.08 Up Tryparedoxin peroxidase (TRYP)
41 LinJ12_v4.0050 2.03 Up Hypothetical protein, conserved
42 LinJ29_V3.0510 2.28 Up Hypothetical protein, unknown function
43 LinJ15_V3.0990 2.09 Up Calmodulin-like protein
44 LinJ29_V3.0910 2.07 Up Hypothetical protein, conserved
45 LinJ26_V3.0730 2.19 Up Hypothetical protein, conserved
Parasitol Res (2014) 113:1171–1184 1177
Table 3 (continued)
S. no. Gene name Fold change[R] versus [S]
Regulation Gene description
46 LinJ23_V3.0590 2.13 Up Hypothetical protein
47 LinJ29_V3.1940 3.65 Up Tobb55,putative
48 LinJ16_V30930 2.20 Up Flagellar calcium-binding protein, putative
49 LinJ18_V3.0890 2.10 Up rab7 GTP binding protein, putative (Rab7)
50 LinJ26_V3.1360 2.32 Up Prefoldin-like protein
51 LinJ13_V3.1400 2.48 Down Chaperonin TCP20, putative
52 LinJ19_V3.1380 2.15 Down Phosphatidic acid phosphatase protein-like protein
53 LinJ22_V3.0590 2.53 Down NADH-cytochrome b5 reductase, putative
54 LinJ06_V3.0370 2.48 Down Glutamine synthetase, putative
55 LinJ10_V3.0660 2.17 Down Endonuclease G, putative
56 LinJ07_V3.0040 2.06 Down Hypothetical protein, conserved
57 LinJ36_V3.1420 2.23 Down Transitional endoplasmic reticulum ATPase, putative, valosin-containing protein homolog
58 LinJ06_V3.0960 2.47 Down 2,4-dienoyl-coa reductase-like protein
59 LinJ04_V3.0280 2.49 Down Hypothetical protein, conserved
60 LinJ25_V3.1040 2.80 Down Hypothetical protein, conserved
61 LinJ36_V3.0250 2.38 Down Peptidyl-prolyl cis-trans isomerase, putative
62 LinJ06_V3.1070 2.64 Down Kinesin, putative
63 LinJ34_V3.1530 2.22 Down Tyrosine phosphatase isoform, putative
64 LinJ25_V3.1790 2.38 Down Pyruvate dehydrogenase E1 beta subunit, putative
65 LinJ06_V3.0290 2.71 Down Ribonuclease H1, putative
66 LmjF22.1290 2.03 Down Ribonucleoside-diphosphate reductase small chain, putative
67 LinJ28_V3.2460 2.21 Down Hypothetical protein, conserved
68 LinJ24_V3.0460 2.48 Down Hypothetical predicted transmembrane protein
69 LinJ25_V3.1190 2.07 Down Hypothetical protein, conserved
70 LinJ21_V3.1330 2.00 Down T-complex protein 1, delta subunit, putative
71 LinJ36_V3.0230 2.44 Down SET domain protein, putative
72 LinJ28_V3.1550 2.34 Down DNA polymerase kappa, putative
73 LinJ28_V3.1180 2.40 Down Hypothetical protein, conserved
74 LinJ34_V3.3710 2.01 Down Hypothetical protein, conserved
75 LinJ32_V3.1970 2.02 Down Hypothetical protein, conserved
76 LinJ06_V3.1350 2.57 Down Hypothetical protein, unknown function
77 LinJ22_V3.0002 2.33 Down Hypothetical protein
78 LinJ36_V3.6980 2.81 Down Hypothetical protein, conserved
79 LmjF06.1290 2.30 Down Hypothetical protein, unknown function
80 LinJ18_V3.0300 2.13 Down Hypothetical protein, conserved
81 LinJ35_V3.4630 2.49 Down Hypothetical protein, conserved
82 LinJ19_V3.1380 2.15 Down Phosphatidic acid phosphatase protein-like protein
83 LinJ35_V3.0070 2.03 Down Prohibitin, putative
84 LinJ04_V3.0940 2.72 Down Chaperone protein DNAj, putative
85 LinJ13_V3.0090 4.18 Down Carboxypeptidase, putative, metallo-peptidase, Clan MA(E), family M 32
Carbohydrate metabolic process GO:0005975
86 LinJ14_V3.0180 3.25 Down Carboxypeptidase, putative, metallo-peptidase, Clan MA(E), family M32
87 LinJ23_V3.1060 2.24 Up Beta-fructosidase-like protein, invertase-like protein, sucrose hydrolase-like protein
88 LinJ30_V3.0170 2.10 Down 2-hydroxy-3-oxopropionate reductase, putative
Lipid metabolic process GO:0006629
89 LinJ31_V3.0870 3.23 Up Lipase precursor-like protein
90 LinJ31_V3.2540 2.60 Up Lipase, putative
91 LinJ14_V3.0700 2.64 Down Fatty acid elongase, putative
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which included ABC1 (LinJ02_V3.0270) and A7(LinJ15_V3.0800) transporter. Aquaglyceroporin (AQP1)transporter (LinJ31_V3.0030) was upregulated in miltefosineresistance which is contrary to observation in antimony resis-tance where AQP1 has been reported to be downregulated(Ouellette et al. 2004). Golgi vesicular membrane trafficking(LinJ29_V3.0660) protein was also upregulated in MIL-RL. donovani. Gene coding for a surface antigen-like proteinand Rab7 GTP binding protein (LinJ18_V3.0890) involved inendocytic pathway were preferentially expressed in MIL-Rparasite. SHERP (LinJ23_V3.1210) and HASPA1, involvedin protein import in organelles and recruitment of anioniclipids were found upregulated in MIL-R parasite, whereas afew transporters including ATPase beta subunit and mitochon-drial carrier protein-like protein were downregulated in MIL-R parasite.
Signal transduction
Signal transduction and cell cycle progression molecules up-regulated in miltefosine resistance included calpain familycysteine protease-like protein (LinJ25_V3.1540), proteinphosphatase 2C (LinJ15_V3.0170), phosphatidic acidphosphatase-like protein (LinJ19_V3.1380), protein kinases[(protein kinase A and serine/threonine protein kinase sos2)LinJ22_V3.0630], and cAMP-specific phosphodiesterase(LinJ15_V3.1540).
Cytoskeleton and motor proteins
Genes coding for a dynein light chain (LinJ05_V3.0070) andkinesin-like protein (LinJ05_V3.0760) were upregulated inMIL-R parasite while a delta-tubulin gene [(d-tub)
Table 3 (continued)
S. no. Gene name Fold change[R] versus [S]
Regulation Gene description
Transporter activity [GO:0006810, GO:0005215]
92 LinJ27_V3.0940 2.53 Up ABCA7, vesicular-fusion ATPase-like protein, putative
93 LinJ06_V3.0090 2.00 Up ATP-binding cassette protein subfamily G, member 5, putative (ABCG5)
94 LinJ05_V3.0810 2.52 Up Hypothetical protein, conserved
95 LinJ36_V3.5100 2.43 Up Hypothetical protein, conserved
96 LinJ33_V3.1420 2.02 Up QA-SNARE protein putative
97 LinJ15_V3.0800 3.65 Up ATP-binding cassette protein subfamily A, member 7, putative (ABCA7)
98 LinJ29_V3.0660 2.63 Up BET1 Like protein, Golgi vesicular membrane trafficking
99 LinJ36_V3.6490 2.97 Up ADP ribosylation factor 3
100 LinJ11_V3.0550 2.15 Up Amino acid permease/transporter
101 LinJ02_V3.0270 2.90 Up ABC 1 transporter
102 LinJ05_V3.1060 2.58 Up ATPase, putative
103 LinJ29_V3.1530 2.93 Up Clathrin coat assembly protein ap19, putative, sigma adaptin, putative adaptin, putative
104 LinJ31_V3.0030 2.79 Up Aquaglyceroporin, AQP1 transporter
105 LinJ15_V3.0900 2.38 Up Nucleotide sugar transporter, putative
106 LinJ14_V3.0330 2.00 Up Hypothetical protein, amino acid transporter like activity
107 LinJ28_V3.2610 3.53 Down Vacuolar ATP synthase subunit b, putative
108 LinJ36_V3.3250 2.37 Down ATP synthase, putative
109 LinJ32_V3.1180 2.06 Down Mitochondrial carrier protein-like protein
110 LinJ32_V3.2190 2.05 Down ABC transporter-like protein
Cellular component, cell organization and biogenesis [GO:0005759, GO:0016043]
111 LinJ06_V3.0780 2.07 Up Hypothetical protein, conserved
112 LinJ25_V3.0990 2.12 Down Gamma-tubulin
113 LinJ36_V3.5220 2.12 Down Delta tubulin, putative
114 LinJ30_V3.1030 2.19 Down p22 protein precursor, putative
115 LinJ34_V3.4070 2.58 Down Hypothetical protein, conserved
Microtubule-based process GO:0007017
116 LinJ05_V3.0070 2.01 Up Dynein light chain, putative
117 LinJ05_V3.0760 2.20 Up Kinesin-like protein
Parasitol Res (2014) 113:1171–1184 1179
LinJ36_V3.5220] and another kinesin protein were downreg-ulated. As described before, transcripts of calpain familycysteine protease-like protein involved in cytoskeleton remod-eling was upregulated in MIL-R parasite.
Q-PCR for validation of microarray experiments
Based on the biological significance, 14 genes were selectedfor validation by q-RT PCR. Results obtained by qRT-PCRwere consistent with the microarray data for all the genesconfirming the differential gene expression betweenmiltefosine resistant and sensitive parasites (Fig. 4).Importantly, to confirm that these modulations are associatedwith miltefosine resistance and are not due to random varia-tion, the expression of selected genes was validated in anothertwo distinct MIL-R induced L. donovani parasites (MIL-R2and MIL-R3) with respect to their corresponding wild-typeparasites, and the results in all MIL-R induced strains werefound consistent with the microarray data. Representative dataon fold changes in gene expression obtained by microarrayand Q-PCR with two MIL R cell lines is shown in Fig. 4.
Discussion
Introduction of oral drug miltefosine represents an importanttherapeutic advance in the treatment of VL in the Indiansubcontinent; however, the development of resistance remainsa serious threat. To understand the mechanism of miltefosineresistance in L. donovani, we carried out mRNA expressionprofiling using full genomic DNA microarray, a techniquesuccessfully employed to study resistance mechanisms inLeishmania (Ubeda et al. 2008; Leprohon et al. 2009; Singh
et al. 2010). Mining of the Leishmania genome revealed themodulated expression of a number of genes that might playimportant role in rendering resistance to the parasite. Weemployed promastigote stage of the parasite for the study asthere is a good correlation in the sensitivity of promastigoteand amastigote forms towards miltefosine (Seifert et al. 2003;Kumar et al. 2009). There is 99.9 % sequence similaritybetween L. infantum and L. donovani genome that justifiesthe use of this array.
The present study with miltefosine-resistant Leishmaniasuggested that the parasite adopts several strategies to counterthe toxic effect of miltefosine (summarized in Fig. 5). Themajor pathways/ mechanism affected in miltefosine resistanceincluded (1) DNA replication and repair machinery, (2) pro-tein translation process, (3) energy generation mechanism, (4)transporters, and (5) antioxidant defense mechanism. Amongthese, the antioxidant defense mechanism has recently beenreported to be operative in miltefosine resistance observed inclinical isolates (Das et al. 2013).
Altered DNA synthesis and repair mechanism
The expression of genes involved in DNA replication andrepair processes was significantly altered in miltefosine resis-tance. DNA replication licensing factor, a DNA-dependentATPase required for initiation of eukaryotic DNA replication,showed decreased expression which may lead to impairedDNA synthesis. This may be combated by enhanced expres-sion of PCNA as these proteins act as the processivity factorfor DNA polymerase delta that help tomaintain the replicationprocess uninterrupted (Lee et al. 2013). Endo/exonucleaseMre11 (Garcia et al. 2011) and A/G-specific adenineglycosylase (Denver et al. 2003) involved in DNA double-
Fig. 4 Validation of DNAmicroarrays expression data byquantitative real-time PCR (qRT-PCR). The gene expressionprofile of selected genes frommicroarray data (green bars) arecompared to qRT-PCR data intwo distinct induced miltefosine-resistant strains (MIL-R1 and 2).Fold changes in gene expressionfor two of the MIL-R parasites,MIL-R1 (red bar) and MIL-R2(blue bar) with respect to MIL-sensitive strains (MIL-S1 andMIL-S2), are represented here.The qRT-PCR data werenormalized with GAPDH gene
1180 Parasitol Res (2014) 113:1171–1184
strand break repair and in base excision repair, respectively,were downregulated, suggesting that parasite adopts bypassprocess for DNA repair and synthesis. However, resistant cellsmay carry out essential DNA repair with the help of upregu-lated RAD51/dmc1 protein (Le Calvez-Kelm et al. 2012), anonsequence-specific DNA binding protein involved in DNArepair. The above facts indicated the compensatory behaviorof parasite machinery to continue the replication processuninterrupted in miltefosine resistance.
Reduced protein synthesis and degradation
Genes involved in translation, protein folding, and degrada-tion were found downregulated in MIL-R parasites. The find-ings suggested increased half-life of proteins in MIL-R para-site since protein synthesis as well as degradation was down-regulated. Downregulation of isoleucyl-tRNA synthetase tran-script indicated reduced protein synthesis, which may becompensated by low proteolytic activity indicated by reducedexpression of chaperone protein DnaJ and ubiquitin-activatingenzyme E1 involved in proteasome-mediated proteolytic pro-cess. Genes involved in protein phosphorylation pathway,signal transduction, and cell cycle progression, such as proteinphosphatase 2C, protein kinase A, and serine/threonine pro-tein kinase sos2, were upregulated during miltefosine resis-tance. Interestingly, prefoldin chaperone transcript was upreg-ulated in miltefosine resistance suggesting compensation forreduced TCP20 and DnaJ chaperons.
In protozoan parasites, cathepsin L proteases have beenknown to play an important role in the infection, replication,development, and metabolism (Mottram et al. 1998). Theelevated levels of cathepsin L transcript in the current studysuggested its role in miltefosine resistance. In cancer, doxoru-bicin resistance was prevented in the presence of inhibitor tocathepsin L, suggesting that inhibition of this enzyme reversesthe development of drug resistance (Zheng et al. 2009).Targeting this enzyme family in miltefosine resistance maytherefore be one strategy in the development of new VLchemotherapy.
Altered energy generation
We did not observe changes in the key enzymes of glycolyticpathway indicating uninterrupted production of pyruvate; how-ever, downregulated expression of Pyruvate dehydrogenase E1beta subunit, the first enzyme of pyruvate dehydrogenase com-plex (PDC) may lead to reduced production of acetyl CoA. Onthe contrary, the upregulated activity of Acetyl-CoA synthasemay promote synthesis of acetyl CoA to produce energy andelectron carriers. However, in MIL-R parasite, ATP productionmay be affected due to downregulation of ATP synthase andvacuolar ATP synthase subunit B, multisubunit proton pumps(Nishi and Forgac 2002) components of oxidative
phosphorylation pathway. Thus, miltefosine pressure eventuallymay lead to reduced energy generation by oxidative phosphor-ylation and the parasite may have to rely on an alternate sourceof energy. The upregulated expression of both lipase and lipaseprecursor-like protein coupled with downregulation of enzymesin lipid biosynthesis (fatty acid elongase and NADH-cytochrome B5 reductase) indicated that lipid catabolism maybe utilized for energy generation in miltefosine resistance.
Upregulation of transporters
Several membrane modifications were indicated inmiltefosine resistance, at the level of plasma membrane (asdepicted by upregulated expression of ABC transporters andHASPAs) as well as mitochondria (SHERP and Tobb55) andendoplasmic reticulum (SHERP; Fig. 5).
The present data showed upregulated expression of ABC1transporters (ABCA7, ABCG5, and ABCG2) in miltefosineresistance. Antimonial resistance was associated with overex-pression of ABC efflux pumps P-glycoproteins (Legare et al.2001) in Leishmania. ABCG-like transporter has a role in drugresistance and transbilayer lipid movement (Castanys-Munozet al. 2007), and its overexpression was shown to affect phos-pholipid trafficking (Castanys-Munoz et al. 2008; Vasiliou et al.2009). An inhibitor of ABC transporters may allow accumula-tion of miltefosine in the parasite and may serve as one of thepossible strategies to combat miltefosine resistance. Proteinkinases A or C have been demonstrated to phosphorylateABC transporters, including P-glycoprotein (Abe-Dohmaeet al. 2004), and ABCA1 (See et al. 2002; Martinez et al.2003; Yamauchi et al. 2003). Upregulated transcripts of proteinkinases in the present study suggested that these may possiblybe modulating the activity of ABC transporters.
SHERP, a 6.2-kDa small hydrophilic endoplasmicreticulum-associated protein, is exclusively expressed onlyin the metacyclic parasites (Brodin et al. 1992; Flinn andSmith 1992; Coulson et al. 1997). It shows weak associationwith the ER and outer mitochondrial membrane and is expect-ed to be involved in various cellular processes such as proteinimport in organelles and recruitment of anionic lipids duringmetacyclogenesis (Knuepfer et al. 2001). The upregulatedexpression of this gene in MIL-R parasite observed in thisstudy indicates the possibility of increased parasite fitness inresistant strains. Upregulated expression of Tob55 mitochon-drial outer membrane protein in the present study indicatedmodulated translocation of ions and small molecules acrossthe mitochondrial membrane in miltefosine resistant parasites.
Antioxidant defense mechanism
Generation of oxidants has been identified as the primarymechanism of drug-induced cell death. The parasite adoptsvarious defense mechanisms to cope with drug-induced
Parasitol Res (2014) 113:1171–1184 1181
oxidative stress by expressing antioxidant proteins such asHSP70 (Brochu et al. 2004) and HSP83 (Vergnes et al.2007) that decrease superoxide radical production and therebythe cell damage. Upregulated expression of prefoldin chaper-one family observed here may likewise be contributing inparasites’ defence against miltefosine pressure (Martin-Benito et al. 2002). Downregulation of prohibitin, a moleculeinvolved in Leishmania–host interaction, observed in the pres-ent study indicated that the MIL-R cells may survive bysuppressing the expression of pro-apoptotic proteins (Jainet al. 2010; Welburn and Murphy 1998).
The ability of trypanosomatids to transform glutathione(GSH) into trypanothione (T[SH]2) is critical for vitality andvirulence of the parasite. T(SH)2, known to enhance anti-oxidant metabolism in Leishmania during drug resistance(Miller et al. 2000; Oza et al. 2003), was found upregulated inMIL-R parasite. Trypanothione synthetase (TrS), a rate-limitingenzyme in the conversion of GSH to T[SH]2, was upregulatedin MIL-R parasite. T[SH]2 has been reported to play a role inantimony resistance inLeishmania (Oza et al. 2003; Goyeneche-
Patino et al. 2008) and the current data suggested its role inresistance towards miltefosine. In Leishmania, T[SH]2 providesreducing equivalents to facilitate the parasite’s defence againstoxidative stress with enzymes of the tryparedoxin peroxidase(TryP) family principally responsible for detoxification of per-oxides (Fairlamb and Cerami 1992; Flohe et al. 1999).Enhanced antioxidant defences indicated by elevated levels ofTryP were observed in antimony and arsenite resistantLeishmania parasites (Lin et al. 2005; Wyllie et al. 2010).Upregulated expression of TryP, TrS, and T[SH]2 in MIL-Rparasite implied that enhanced antioxidant defences, may wellbe a key feature of resistance to miltefosine.
In conclusion, MIL-R parasite uses alternative strategiesfor uninterrupted DNA replication, protein synthesis andproper folding, meeting energy demand of the cell, transportof ions and small molecules, and antioxidant defense mecha-nism to subvert the drug effects. The study contributes to-wards understanding perturbed biochemical behavior ofLeishmania in response to high miltefosine pressure. Theinterdependent regulation of gene expression to combat the
Fig. 5 Model depicting mechanism of miltefosine resistance inL. donovani. Genes altered in MIL-R parasite are represented. Genesmarked with arrow represent the upregulated genes and the ones markedwith arrow represent the downregulated genes in case of MIL-R parasite.1, 2, 3, 4, 5, and 6 The probable mechanisms of resistance in the MIL-Risolate. 1 ABC 1 transporters (ABCA1 and A7) upregulated in MIL-Rthat lead to efflux of MIL out of the cell. Protein kinase (PK), known tophosphorylate the ABC1 transporters, stabilize the expression of theseproteins on plasma membrane, possibly contributing to MIL-R. 2Lipasesupregulated inMIL-R are involved in fatty acid metabolism and free fattyacids (FFA) from lipid degradation could be destined to beta oxidation forenergy generation as an alternate energy source. 3 Upregulation of
AcetylcoA synthetase (involved in TCA cycle) in MIL-R. 4Upregulationof trypanothione [T(SH)2] biosynthesis by trypanothione synthetase(TrS) in MIL-R enhances the anti-oxidant metabolism of Leishmaniaparasites thereby contributing to MIL resistance. Upregulation oftryparedoxin peroxidase [TP(ox)], responsible for hydroperoxide detox-ification further aids in antioxidant defense in MIL-R. 5 Altered DNAsynthesis indicated by decreases expression of DNA replication licensingfactor may be combated by increased expression of PCNA and DNArepair enzymes RAD51/dmc1. 6Reduced protein translation, folding, anddegradation indicated by reduced expression of aminoacyltRNAsynthatase, cheparon proteins, and ubiquitin activating enzymes
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drug pressure indicates that development of parasite’s resis-tance towards a drug is multifactorial phenomenon that helpsparasite to sustain the lethal effects of miltefosine. Suchknowledge could be helpful in monitoring resistance in clin-ical isolates or to subsequently target major molecular inter-actions associated with miltefosine resistance.
Acknowledgments We are thankful to Dr Marc Ouellette at the Re-search Centre in Infectious Diseases, Faculty of Medicine, Laval Univer-sity, Quebec, Canada for kindly sharing the microarray design with us.AK and VS are grateful to Council for Scientific and Industrial Researchand University Grants Commission, India, respectively, for providingresearch fellowship. This work was supported by Indian Council ofMedical Research grant number 63/4/2007/-BMS.
Conflict of interest The authors do not have commercial or otherassociations that might pose any competing interest.
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