Parasitol Res (2014) 113:1171–1184 DOI 10.1007/s00436-014-3755-6
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 subcontinent. Oral miltefosine therapy has recently replaced antimonials in endemic areas. However, the drug is at risk of emergence 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 miltefosineresistant 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 mechanisms by which the parasite sustains miltefosine pressure including (1) compromised DNA replication/repair mechanism, (2) reduced protein synthesis and degradation, (3) altered 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
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
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 leishmaniasis (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 selective activity against Leishmania (Clive et al. 1999; Croft et al. 2006). In India, miltefosine has recently taken over as the firstline 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 emergence of resistance in India, where VL is anthroponotic (Bryceson 2001). The long half-life of the drug may potentially increase the risk of development of experimental resistance 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 understand the mechanism of action and development of resistance towards miltefosine in the parasite. The mechanisms and related biological pathways that contribute 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
1172
transduction as well as inhibition of acyl transferase, an enzyme involved in lipid remodeling (Lux et al. 2000). Current evidence suggests that the drug kills Leishmania cells by a process reminiscent of programmed cell death (Verma and Dey 2004; do Monte-Neto et al. 2011). An impairment in drug uptake machinery involving amino-phospholipid translocase miltefosine transporter (LdMT) and an accessory protein, LdRos3 (CDC50/Lem3 family) in experimental miltefosine-resistant Leishmania lines was proposed to be the most likely mechanism of resistance (Perez-Victoria et al. 2006). Proteomic analysis revealed role of eukaryotic initiation factor eIF4 in miltefosine resistance in L. donovani (Singh et al. 2008). Regardless, a better understanding of the molecular mechanism involved is of paramount importance. The completion of the genomic sequences of several Leishmania species (http://www.genedb.org/) provided the opportunity to study the pattern of whole-genome differential expression during drug resistance. In recent times, gene expression microarray has become a well-established technology by which the expression of thousands of genes can be measured simultaneously providing a global genetic perspective on complex biological processes like drug resistance. Various studies have demonstrated the usefulness of wholegenome DNA microarrays for studying drug resistance in Leishmania (Ubeda et al. 2008; Leprohon et al. 2009; Singh et al. 2010). However, the modulations in Leishmania transcriptome in miltefosine resistance are poorly explored. The current study utilized whole-genome Leishmania spp. oligonucleotide array to explore differences in gene expression between miltefosine resistant and sensitive L. donovani parasite. In this study, populations of L. donovani resistant to miltefosine were selected in vitro in order to study global gene expression modulation associated with resistance. In-depth bioinformatic analysis was performed to identify changes in groups of interacting genes or pathways that may contribute to resistance to miltefosine. We have found evidence of altered expression of several genes belonging to DNA repair and replication machinery, protein translation and folding, energy generation by lipid degradation, transporter activity, and antioxidant defense mechanism in miltefosine-resistant L. donovani parasite.
Parasitol Res (2014) 113:1171–1184
lines referred as MIL-S1 MIL-S2 and MIL-S3) were cultured as promastigotes in Medium 199 (Sigma Aldrich, USA), 25 mM HEPES N-[2-hydroxyethyl]piperazine-N −1 -[2ethanesulfonic acid] (Sigma Aldrich, USA), 100 IU, and 100 μg/ml each of penicillin G (Sigma Aldrich, USA) and streptomycin 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 were characterized as L. donovani by species-specific PCR (Salotra et al. 2001). Preparation of miltefosine stock/drug stock Miltefosine (Cayman Chemical Company, USA) stock was prepared by dissolving the drug at concentration of 5 mg/ml in absolute methanol and stored at 4 °C up to 1 month. The working stock was prepared fresh in Medium 199 on the day of experiment. Generation and characterization of experimental L. donovani strains resistant to miltefosine
Materials and methods
Wild-type parental L. donovani lines (MIL-S1, MIL-S2, and MIL-S3) were adapted to grow under high MIL pressure by in vitro passage with a stepwise increase in the miltefosine concentration (2.5, 5, 7.5,10, 20, and 30 μg/ml) in medium M199 to generate miltefosine-resistant parasite designated as MIL-R1, MIL-R2, and MIL-R3. At each step, parasites were cultured for at least five to eight passages to attain steady and optimal cell growth comparable to its wild-type miltefosinesensitive counterpart. The MIL-R1 line was taken up further for microarray analysis, while validation of microarray data by real-time PCR was done in three miltefosine-resistant line (MIL-R1, R2, and R3). The susceptibility of miltefosineresistant L. donovani (MIL-R1) to current antileishmanial drugs (miltefosine, SAG, amphotericin B, paromomycin, and sitamaquine) was tested at intracellular amastigote stage as described previously (Singh et al. 2010; Kulshrestha et al. 2011). Single-nucleotide polymorphism, in LdMT and LdRos3 genes, associated with miltefosine resistance was determined for both sensitive and resistant cell lines. The genes were PCR amplified and amplicons were sequenced on Automated Sequencer ABI 3730. Primers used for amplification and sequencing are given as ESM Tables S1.1 and S1.2.
Parasite culture
Oligonucleotide array
Parasite isolates were prepared from bone marrow aspirate of VL patient originating from Bihar and reporting to Safdarjung Hospital (SJH), New Delhi, as described previously (Kulshrestha et al. 2011). Informed consent was obtained from patients according to the guidelines of the Ethical Committee, SJH. This wild-type parental line (miltefosine-sensitive cell
One-color microarray-based gene expression profiling was carried out using a high-density Leishmania multispecies 60-mer oligonucleotide microarray slide [8×15K format] representing the entire genome of Leishmania infantum and Leishmania major. The microarray chip, printed by Agilent Technologies, USA, included a total of 9,233 Leishmania-specific genes
Parasitol Res (2014) 113:1171–1184
including 540 control probes. Gene expression analysis employing this array has been described previously (Ubeda et al. 2008; Leprohon et al. 2009; Rochette et al. 2008). RNA isolation Total RNA was extracted from 10 8 late log phase promastigotes using Trizol reagent according to manufacturer’s instructions. RNA clean up was performed using RNeasy Plus mini kit (Qiagen, Gaithersburg, MD, USA) as described by the manufacturer. The purified RNA was quantified using Nanodrop by estimating the absorbance at 260 and 280 nm. The quality and integrity of RNA was assessed on RNA 6000 Nano Assay Chips on Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The presence of three distinct ribosomal peaks (18S, 24Sα, and 24Sβ) confirmed successful RNA extraction. RNA labelling and microarray hybridization Complementary RNA (cRNA) was generated from 1 μg of total RNA using Quick-Amp Labeling kit (Agilent Technologies) that directly incorporates Cy-3 labeled CTP into the cRNA. Prehybridization and hybridization were performed according to manufacturers’ instructions. Labeled probes were hybridized with Leishmania oligonucleotide array using Gene Expression Hybridization Kit (Agilent Technologies) at 65 °C for 17 h. The hybridized arrays were subsequently washed with gene expression hybridization buffers 1 and 2 using 0.005 % Triton X-102. Experiments were performed using three independent RNA extractions. The slides were scanned immediately in Axon GenePix 4000B scanner to minimize the impact of environmental oxidants on signal intensities. GeneSpring GX 11.0.2 microarray data and pathway analysis tool was used for data analysis. Analysis involved data preprocessing; elimination of outliers, nonsignificantly expressed genes and false positives; and analysis of the gene lists in a biological perspective. The data files were in text (.txt) format and obtained from Agilent’s Feature Extraction (FE). The summarization of the “raw” signal values was performed by computing the geometric mean. “Normalized” value was generated after log transformation and normalization (scale) and baseline transformation. For each probe, the median of the log summarized values from all the samples is calculated and subtracted from each of the samples. Quartile (75th percentile) normalization was performed. Storey and bootstrapping analysis was performed for multiple testing corrections. Statistically significant differentially expressed genes were determined by t test (unpaired) for two groups; (p value cutoff)30c ND 17.76±1.38 0.81±0.18 0.52±0.14 6.14±0.29 0.91±0.15 19.64±1.35 4.70±0.09
0.0003*
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 subcontinent. Oral miltefosine therapy has recently replaced antimonials in endemic areas. However, the drug is at risk of emergence 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 miltefosineresistant 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 mechanisms by which the parasite sustains miltefosine pressure including (1) compromised DNA replication/repair mechanism, (2) reduced protein synthesis and degradation, (3) altered 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
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
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 leishmaniasis (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 selective activity against Leishmania (Clive et al. 1999; Croft et al. 2006). In India, miltefosine has recently taken over as the firstline 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 emergence of resistance in India, where VL is anthroponotic (Bryceson 2001). The long half-life of the drug may potentially increase the risk of development of experimental resistance 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 understand the mechanism of action and development of resistance towards miltefosine in the parasite. The mechanisms and related biological pathways that contribute 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
1172
transduction as well as inhibition of acyl transferase, an enzyme involved in lipid remodeling (Lux et al. 2000). Current evidence suggests that the drug kills Leishmania cells by a process reminiscent of programmed cell death (Verma and Dey 2004; do Monte-Neto et al. 2011). An impairment in drug uptake machinery involving amino-phospholipid translocase miltefosine transporter (LdMT) and an accessory protein, LdRos3 (CDC50/Lem3 family) in experimental miltefosine-resistant Leishmania lines was proposed to be the most likely mechanism of resistance (Perez-Victoria et al. 2006). Proteomic analysis revealed role of eukaryotic initiation factor eIF4 in miltefosine resistance in L. donovani (Singh et al. 2008). Regardless, a better understanding of the molecular mechanism involved is of paramount importance. The completion of the genomic sequences of several Leishmania species (http://www.genedb.org/) provided the opportunity to study the pattern of whole-genome differential expression during drug resistance. In recent times, gene expression microarray has become a well-established technology by which the expression of thousands of genes can be measured simultaneously providing a global genetic perspective on complex biological processes like drug resistance. Various studies have demonstrated the usefulness of wholegenome DNA microarrays for studying drug resistance in Leishmania (Ubeda et al. 2008; Leprohon et al. 2009; Singh et al. 2010). However, the modulations in Leishmania transcriptome in miltefosine resistance are poorly explored. The current study utilized whole-genome Leishmania spp. oligonucleotide array to explore differences in gene expression between miltefosine resistant and sensitive L. donovani parasite. In this study, populations of L. donovani resistant to miltefosine were selected in vitro in order to study global gene expression modulation associated with resistance. In-depth bioinformatic analysis was performed to identify changes in groups of interacting genes or pathways that may contribute to resistance to miltefosine. We have found evidence of altered expression of several genes belonging to DNA repair and replication machinery, protein translation and folding, energy generation by lipid degradation, transporter activity, and antioxidant defense mechanism in miltefosine-resistant L. donovani parasite.
Parasitol Res (2014) 113:1171–1184
lines referred as MIL-S1 MIL-S2 and MIL-S3) were cultured as promastigotes in Medium 199 (Sigma Aldrich, USA), 25 mM HEPES N-[2-hydroxyethyl]piperazine-N −1 -[2ethanesulfonic acid] (Sigma Aldrich, USA), 100 IU, and 100 μg/ml each of penicillin G (Sigma Aldrich, USA) and streptomycin 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 were characterized as L. donovani by species-specific PCR (Salotra et al. 2001). Preparation of miltefosine stock/drug stock Miltefosine (Cayman Chemical Company, USA) stock was prepared by dissolving the drug at concentration of 5 mg/ml in absolute methanol and stored at 4 °C up to 1 month. The working stock was prepared fresh in Medium 199 on the day of experiment. Generation and characterization of experimental L. donovani strains resistant to miltefosine
Materials and methods
Wild-type parental L. donovani lines (MIL-S1, MIL-S2, and MIL-S3) were adapted to grow under high MIL pressure by in vitro passage with a stepwise increase in the miltefosine concentration (2.5, 5, 7.5,10, 20, and 30 μg/ml) in medium M199 to generate miltefosine-resistant parasite designated as MIL-R1, MIL-R2, and MIL-R3. At each step, parasites were cultured for at least five to eight passages to attain steady and optimal cell growth comparable to its wild-type miltefosinesensitive counterpart. The MIL-R1 line was taken up further for microarray analysis, while validation of microarray data by real-time PCR was done in three miltefosine-resistant line (MIL-R1, R2, and R3). The susceptibility of miltefosineresistant L. donovani (MIL-R1) to current antileishmanial drugs (miltefosine, SAG, amphotericin B, paromomycin, and sitamaquine) was tested at intracellular amastigote stage as described previously (Singh et al. 2010; Kulshrestha et al. 2011). Single-nucleotide polymorphism, in LdMT and LdRos3 genes, associated with miltefosine resistance was determined for both sensitive and resistant cell lines. The genes were PCR amplified and amplicons were sequenced on Automated Sequencer ABI 3730. Primers used for amplification and sequencing are given as ESM Tables S1.1 and S1.2.
Parasite culture
Oligonucleotide array
Parasite isolates were prepared from bone marrow aspirate of VL patient originating from Bihar and reporting to Safdarjung Hospital (SJH), New Delhi, as described previously (Kulshrestha et al. 2011). Informed consent was obtained from patients according to the guidelines of the Ethical Committee, SJH. This wild-type parental line (miltefosine-sensitive cell
One-color microarray-based gene expression profiling was carried out using a high-density Leishmania multispecies 60-mer oligonucleotide microarray slide [8×15K format] representing the entire genome of Leishmania infantum and Leishmania major. The microarray chip, printed by Agilent Technologies, USA, included a total of 9,233 Leishmania-specific genes
Parasitol Res (2014) 113:1171–1184
including 540 control probes. Gene expression analysis employing this array has been described previously (Ubeda et al. 2008; Leprohon et al. 2009; Rochette et al. 2008). RNA isolation Total RNA was extracted from 10 8 late log phase promastigotes using Trizol reagent according to manufacturer’s instructions. RNA clean up was performed using RNeasy Plus mini kit (Qiagen, Gaithersburg, MD, USA) as described by the manufacturer. The purified RNA was quantified using Nanodrop by estimating the absorbance at 260 and 280 nm. The quality and integrity of RNA was assessed on RNA 6000 Nano Assay Chips on Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The presence of three distinct ribosomal peaks (18S, 24Sα, and 24Sβ) confirmed successful RNA extraction. RNA labelling and microarray hybridization Complementary RNA (cRNA) was generated from 1 μg of total RNA using Quick-Amp Labeling kit (Agilent Technologies) that directly incorporates Cy-3 labeled CTP into the cRNA. Prehybridization and hybridization were performed according to manufacturers’ instructions. Labeled probes were hybridized with Leishmania oligonucleotide array using Gene Expression Hybridization Kit (Agilent Technologies) at 65 °C for 17 h. The hybridized arrays were subsequently washed with gene expression hybridization buffers 1 and 2 using 0.005 % Triton X-102. Experiments were performed using three independent RNA extractions. The slides were scanned immediately in Axon GenePix 4000B scanner to minimize the impact of environmental oxidants on signal intensities. GeneSpring GX 11.0.2 microarray data and pathway analysis tool was used for data analysis. Analysis involved data preprocessing; elimination of outliers, nonsignificantly expressed genes and false positives; and analysis of the gene lists in a biological perspective. The data files were in text (.txt) format and obtained from Agilent’s Feature Extraction (FE). The summarization of the “raw” signal values was performed by computing the geometric mean. “Normalized” value was generated after log transformation and normalization (scale) and baseline transformation. For each probe, the median of the log summarized values from all the samples is calculated and subtracted from each of the samples. Quartile (75th percentile) normalization was performed. Storey and bootstrapping analysis was performed for multiple testing corrections. Statistically significant differentially expressed genes were determined by t test (unpaired) for two groups; (p value cutoff)30c ND 17.76±1.38 0.81±0.18 0.52±0.14 6.14±0.29 0.91±0.15 19.64±1.35 4.70±0.09
0.0003*
