J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

ScienceDirectwww.e l sev i e r . com/ loca te / j p ro t

A quantitative proteomic screen to identify

potential drug resistance mechanism inα-difluoromethylornithine (DFMO)resistant Leishmania donovani

Alok Kumar Singha, Sigrid Robertsb, Buddy Ullmanc, Rentala Madhubalaa,⁎aSchool of Life Sciences, Jawaharlal Nehru University, New Delhi, IndiabSchool of Pharmacy, Pacific University, OR, USAcDepartment of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, USA

A R T I C L E I N F O

⁎ Corresponding author at: School of Life ScieE-mail address: rentala@outlook.com (R.

http://dx.doi.org/10.1016/j.jprot.2014.02.0301874-3919/© 2014 Elsevier B.V. All rights rese

A B S T R A C T

Article history:Received 27 August 2013Accepted 25 February 2014

Visceral leishmaniasis (VL) caused by Leishmania donovani is a systemic protozoandisease that isfatal if left untreated. The promastigote form of L. donovani is sensitive to growth inhibition byDL-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the firstenzyme of the polyamine biosynthetic pathway. Exposure of a wild type (DI700) cell populationto gradually increasing concentrationsofDFMOresulted in the selection of a strain of Leishmania(DFMO-16),whichwas capable of proliferating in 16 mMDFMO.To elucidate themolecular basisfor this resistance,weundertook a comparative proteomic analysis of DFMO-resistant/sensitiveparasites using isobaric tagging for relative and absolute quantification (iTRAQ/LC–MS/MS). Outof the 101 proteins identified in at least 2 of the 3 independent experiments, 82 proteins are 1.5-to 44.0-fold more abundant in DFMO-resistant strain (DFMO-16) while 19 are 2- to 5.0-fold lessabundant as compared to the wild-type (DI700) parasites. Proteins with 2-fold or greaterabundance in the DFMO-resistant strain include free radical detoxification, polyamine andtrypanothione metabolic proteins, proteins involved in metabolism, intracellular survival andproteolysis, elongation factors, signalingmolecules andmitochondrial transporters, and manywith no annotated function. Differentiallymodulated proteins contribute to our understandingof molecular mechanism of DFMO-resistance and have the potential to act as biomarkers.

Biological significanceThis study will facilitate a deeper understanding of the phenomenon of acquired drugresistance and possible biomarkers in Leishmania against antiparasitic drug DFMO. Also itwill provide information about the metabolic pathways modulated in resistant parasites asan adaptation mechanism to counter drugs. Studies like this are important to safeguard theefficacy of a limited repertoire of anti-parasitic drugs, and to lead the development of newdrugs and drug combinations.

© 2014 Elsevier B.V. All rights reserved.

Keywords:Leishmania donovaniPolyaminesDrug resistanceDifluoromethylornithineProteomicsOrnithine decarboxylase

nces, Jawaharlal Nehru University, New Delhi 110067, India. Tel./fax: +91 11 26742630.Madhubala).

rved.

45J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

1. Introduction

Visceral leishmaniasis (VL) is caused by the protozoan parasiteLeishmania donovani and is fatal if left untreated. Due to the lack ofan effective vaccine and the problems related to vector control,chemotherapy has offered the only avenue for treating thisdisease [1]. Pentavalent antimony (SbV) remains the cornerstoneand first line of drug against VL in almost all regions of theworld[2]. Due to emergence of antimony-resistance in several parts ofthe world and most notably in India, there is a need to look fornewer alternatives [1].

Polyamines play an essential role in macromolecular bio-synthesis and cell proliferation [3]. The polyamine pathway hasbeen targeted in a multiplicity of antineoplastic and antipara-sitic drug regimens [4]. The polyamine derivative trypanothione(T[SH]2) in trypanosomatids is responsible for defense againstoxidative stress and maintaining intracellular redox equilibri-um [5]. Trypanothione is synthesized by trypanothione synthe-tase by conjugating two glutathione molecules to a spermidinemolecule [6]. L-Arginine, an essential amino acid is the precur-sor of polyamines where its enzymatic hydrolysis by arginaseresults in the formation of L-ornithine and urea. Ornithinedecarboxylase (ODC), a homodimeric enzyme converts L-orni-thine into putrescine by an enzymatic decarboxylation [7]. Theessentiality of enzymes in polyamine biosynthesis pathway hasbeen investigated in detail by generating null mutants of thegenes encoding these enzymes in trypanosomes and Leishmania[8–11].

DL-α-Difluoromethylornithine (DFMO) is anenzyme-activated,irreversible inhibitor of ornithine decarboxylase (ODC), involvedin the rate-limiting step in polyamine biosynthetic pathway[12]. Lower level of spermidine in DFMO treated parasites hasinhibitory effect on trypanothione pool and is detrimental toparasite growth and proliferation [13]. DFMO has emerged as apromising drug for selective eradication of parasitic infectionand has shown considerable efficacy in treating African sleepingsickness caused by Trypanosoma brucei gambiense [14]. It hasalso been successfully used against other trypanosomes andhas shown considerable cytotoxicity against Leishmania [15,16].Selective anti-parasitic effect of DFMO for themetabolic machin-ery of the parasite is not due to differential sensitivities of theparasite andhumanODCenzymes to inactivationbyDFMObut israther due to a novel mechanism involving disparities in ODCturnover rates between T. brucei and the mammalian host [17].L. donovani refractory to α-DFMO shows characteristic geneamplification of ODC, resulting in an elevated level of ODCexpression and activity [18]. Clinical isolates of T. bruceirhodesiense show 50–70% lower DFMO uptake as compared tosensitive strains but ODC activities, spermidine and spermineuptake rates, polyamine content and inhibition of polyaminemetabolism by DFMO were statistically similar between sensi-tive and refractory isolates. Previous studies indicate a signif-icantly altered S-adenosylmethionine (S-AdoMet) metabolismin DFMO-resistant isolates indicating engagement of AdoMetmetabolism as a principal axis in DFMO-resistance in parasites[19].

However recent reports of clinical isolates of trypanosomesrefractory to DFMO treatment advocate an early need to in-vestigate the active principles and the molecular mechanism

underlying this resistance. Previous reports based on geneknockouts, over-expression of enzymes and use of specificinhibitors like DFMO and its analogues while validate theessentiality of the polyamine biosynthetic pathway; howeverfail to provide a key insight into criticalmetabolic changes at theglobal level in the parasites. Drug-resistant strains have beenexamined at the DNA sequence level, but seldom usinglarge-scale quantitative proteomics. In the present study, wehave compared the proteome of the DFMO-resistant strain(DFMO-16) with the proteome of the drug-sensitive strain(DI700).

To track proteome level changes in laboratory inducedDFMO-resistant L. donovani (DFMO-16) as compared to its wildtype parental control (DI700) we employed isobaric taggingmethodology, isobaric tags for relative and absolute quantifi-cation (iTRAQ) followed by high resolution mass spectroscopy(MS). Using stationary phase infective promastigotes, we ob-served major changes in metabolic pathways where tricar-boxylic acid cycle (TCA) and sulfur-containing amino acidpathways were distinctly modulated in providing criticalreaction intermediates fueling polyamine biosynthesis. Weobserved abundance among a series of survival factors likeparasitic proteases, elongation factors and heat shock pro-teins implicated in host–parasite interaction, oxidant resis-tance and apoptosis. Proteins involved in GTPase mediatedsignaling, vesicular trafficking and nucleotide transportwere critically modulated suggesting importance of alteredsignaling events and energy metabolism. These observationssuggest that Leishmania promastigotes resistant to DFMOreprogram their metabolic pathways and intracellular surviv-al factors that are crucial during infection and thus relevantfor regulation of parasitic virulence.

2. Materials and methods

2.1. Parasite and culture conditions

Thewild type strainused inall experimentswas theDI700 cloneof the 1S Sudanese strain of L. donovani. Promastigotes of L.donovani (DI700) were routinely cultured inmodified completelydefined culture medium DME-L that contains Dulbecco'smodified Eagle's medium as the powder base and is supple-mented with 5 mg/l of hemin and 0.1 mM of xanthine [18].Exposure of wild type (D1700) cell population to graduallyincreasing concentrations of DFMO resulted in the selection of astrain of Leishmania, DFMO-16, which was capable of proliferat-ing in 16 mM DFMO. Promastigotes of DFMO-resistant strain(DFMO-16) were propagated in a completely defined culturemedium DME-L and 16 mM DFMO (difluoromethylornithine;Sigma, St. Louis, MO, USA) for over 1000 generations [20]. ForiTRAQ analyses, stationary phase parasites of both strains weregrown for two successive generations. Fig. 1 shows the flowchart depicting iTRAQ methodology used in the current study.Three individual promastigote cultures were maintained forboth wild type (DI700) and DFMO-resistant (DFMO16) in parallelfor obtaining protein and RNA. We used metacyclic promas-tigotes that were maintained up to 5 days in culture. Threeindividual culture samples for DI700 and DFMO-16 were con-sidered as three biological replicates.

Fig. 1 – Flowchart illustrating the iTRAQ workflow used to quantify the differential proteome in α-difluoromethylornithine(DFMO) resistant Leishmania donovani. Quantitative real-time PCR was used to validate expression level of gene transcriptscorresponding to selected proteins that were significantly modulated.

46 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

2.2. Protein extraction

Metacyclic promastigotes were harvested and washed thricewith ice cold phosphate buffered saline (PBS). Parasites werelysed in membrane lysis buffer (Invitrogen, Carlsbad, CA,USA). Briefly, Halt Protease Inhibitor Cocktail (Thermo FisherScientific Inc., USA), phenylmethylsulfonyl fluoride (Sigma,St. Louis. MO, USA) and nuclease inhibitor benzonase (Sigma,St. Louis, MO, USA), were added to the lysis buffer. Cell lysatewas centrifuged at 13,000 rpm for 20 min at 4 °C and super-natant was collected. Protein estimation was carried out usingBradford method [21].

2.3. iTRAQ labeling

For iTRAQ labeling equal amount of protein from DI700parental and DFMO-16 strain was acetone precipitated over-night at −20 °C followed by centrifugation at 13,000 rpm for20 min at 4 °C. The iTRAQ labeling was performed accordingto the manufacturer's protocol (Applied Biosystems, FosterCity, CA). Briefly, 100 μg of soluble proteins from each samplewas dissolved in 20 μL of dissolution buffer and 1 μL of dena-turant reagent. The samples were reduced for 1 h at 60 °Cwith 2 μL reducing agent. Cysteine blocking was performedusing 1 μL of cysteine blocking reagent for 10 min at roomtemperature. The tryptic digestion was performed by adding10 μL of trypsin (Sigma, St. Louis, MO, USA) solution (prepared

as 1 μg/μL in water solution with enzyme and substrate ratioof 1:10) followed by incubation for 16 h at 37 °C. The peptideswere labeled using iTRAQ reagent (iTRAQ label 114 for DI700parental, iTRAQ label 116 for DFMO-16) previously constitutedin 75 μL of ethanol for 1 h at 37 °C according to the (iTRAQReagent multiplex) kit protocol. Differentially labeled sampleswere pooled together and further strong cation exchange(SCX) chromatography was carried out.

2.4. SCX fractionation

The labeled peptide mixture was separated by off-line SCXchromatography using HPLC with a UV detector (1200 series,Quaternary pumps, Agilent). The labeled samples (200 μg)were resuspended in SCX low ionic strength buffer (5 mMammonium formate), 30% acetonitrile (ACN) and loaded ontoPolyLC Polysulfoethyl A Zorbax-A SCX column, 5 μm (2.1 mm ×150 mm, Agilent). Peptides were eluted with increasing con-centrations of 5 mM ammonium formate, 30% ACN to 500 mMammonium formate, 30%ACN. 30–35 fractionswere collectedata flow rate of 400 μL/min.

Further offline fractionation for labeled peptides was per-formed on a Tempo nano-LC and spotted on MALDI plates.Three independent biological replicate experiments were per-formed to increase the proteome coverage and reproducibilityof the result. Equal amount of protein (100 μg) from both thewild-type and DFMO-resistant parasites was mixed together

47J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

and subjected to one dimensional chromatography followedby MS analysis to obtain peptide masses.

2.5. Nano-LC and MS analysis

Analysis of the peptide mixture was conducted using liquidchromatography (Tempo nano-LC from Applied Biosystems,Foster City, CA) offline coupled to ABI 5800 Proteomics AnalyzerMALDI-TOF/TOF mass spectrometer (Applied Biosystems,Foster City, CA). Each fraction was dissolved in 15 μL of 1Abuffer (98% water, 2% acetonitrile, ACN and 0.1% trifluoroaceticacid, TFA) and 12 μL of thiswas takenupby an autosampler anddirectly loaded onto LC tempo column (Chromolith Cap RodRP-18e (150 × 0.1 mm) monolithic capillary) and separatedusing 64 min gradient: 5% ACN to 60% ACN. The column elutesweremixedwith 5 mg CHCA (α-cyano-4 hydroxycinnamic acid)matrix in 85% ACN, 0.1% TFA was spotted at a 1.5 μL/min flowrate on 1232 well (44 × 28) LC-MALDI stainless steel plate. ABSCIEX MALDI-TOF/TOF 5800 Analyzer (AB SCIEX, Foster City,CA) equipped with a neodymium: yttrium–aluminum-garnetlaser (laser wavelength used was 349 nm) was used to performprotein identification. The TOF/TOF calibration mixture of ref-erence peptides was used to calibrate the spectrum to a masstolerance with 50 ppm. For MS mode, at least 1500 laser shotswere typically accumulated and 800–400 m/z mass rangewas used per spectrum. The peptides were fragmented withcollision-induced dissociation (CID) energy of 1 kV. For CIDexperiments, ambient air was used as collision gas with me-dium pressure of 10−6 Torr. The 30 most intense precursors perspot were selectedwith aminimumsignal-to noise (s/n) ratio of25 and were fragmented in the CID mode. The peak detectioncriteria used were a minimum s/n of 10, a local noise windowswidth mass/charge (m/z) of 250, and minimum full-widthhalf-maximum (bins) of 2.9. 4000 acquisitions were accumulat-ed for each MS/MS spectrum with dynamic exit mode andiTRAQ capture mode. The interpretation for MS/MS analysisincludes the exclusion of contaminant m/z peaks originatingfrom human keratin, trypsin autodigestion and matrix.

2.6. Data analysis

Data analysis (MS and MS/MS) and database searching wereconducted against theoretical Leishmania infantum version3.0 proteome (http://www.genedb.org/genedb/linfantum) usingProtein Pilot software (version 3; Applied BiosystemsMDS Sciex)with the paragon method utilizing the following search param-eters: L. infantum as species, trypsin as enzyme (two missedcleavage allowed),with fixed modification of methyl methanethiosulfonate (MMTS)-labeled cysteine parameter enabled,ITRAQ (4-plex peptide labeled at N terminus and lysine) assample type. The ‘Search Effort’ parameter Thorough ID' whichprovides a broad search for various protein modifications andmultiple mass cleavages were chosen. The Paragon Algorithmused in Protein Pilot requires no definition of peptide/fragmentmass tolerance, as it iteratively searches for the optimal masserror for a data set. The raw peptide identification results fromthe Paragon Algorithm (Applied Biosystems) searches werefurther processed by Pro Group Algorithm (Applied Biosystems)within the Protein Pilot software before display. The majority ofaverage protein ratios reported by Protein Pilot have a p-value

(evaluating the statistical difference between the observed ratioand unity) and EF (error factor) for each protein ID. The EF termindicates the actual average value that lies between (reportedratio) / (EF) and (reported ratios) × (EF) at 95% confidence.

The parameters that were used for identification andquantification of differentially expressed proteins included:(1) Threshold of 5% accepted Global False Discovery rate(G-FDR) proteins; (2) minimum protein confidence thresholdcut-off was 95% (Unused-ProtScore > 2.0); and (3) at least 1peptide with 95% confidence for the relative abundance [22].In each biological experiment peptides from DFMO-sensitivestrain (DI700) were labeled using isobaric label 114 while label116 was used to label peptides derived from DFMO-16 strain.The ratios of peak areas (116:114) for each of the signatureions {114 (DI700) and 116 (DFMO-16)} respectively from thebiological replicate experiments were obtained. The bias cor-rection algorithm was applied to correct for unequal mixingduring the combination of differently labeled samples. Allquant ratios (both the average ratio for protein and individualpeptide ratios) were corrected for the bias. The false positiverates of the aforementioned filter criteria were all below 5%,estimated by using an individual reversed (decoy) sequencefor the entire Leishmania proteome as described previously[23]. Experiments were performed with three independentbiological replicates and Progroup (Applied Biosystems) soft-ware was used to pool data from all experiments. Proteinsidentified by Protein Pilot software with ≥95% confidence(Unused-ProtScore ≥ 1.3) and iTRAQ ratios ≥1.5 or ≤0.5 wereconsidered differentially abundant. Ratios ≥1.5 were listed asincreased abundance while ratios ≤0.5 were considered asdecreased abundance. The iTRAQ ratios that were consistentbetween two independent experiments with a p-value of lessthan 0.05 were flagged as differentially expressed. All statis-tical tests and analyses were performed using Excel andGraph Pad Prism5 software.

2.7. RNA Preparation and real-time PCR (RT-PCR) analysis

Total RNA was isolated using Tri reagent (Sigma, St. Louis.MO, USA) treated with RNase-free DNase (Invitrogen) as permanufacturer's instruction. Purified RNA (500 ng) was reversetranscribed using First strand cDNA synthesis kit (Fermentas,Germany) according to manufacturer's instruction. Controlshaving the same amount of RNA but lacking reverse tran-scriptase or template were used to rule out DNA or any con-tamination. RT-PCR was performed on the resulting cDNAsusing SYBR Fast Green double-stranded DNA binding dye(Applied Biosystems) and the ABI PRISM 700 SequenceDetection System instrument (Applied Biosystems). The effi-ciency of each PCR was evaluated by performing a 10-folddilution series experiment. Accession number of each gene,the primer sequence, amplicon size, average slope andaverage R2 value for each primer pair are given in theSupplementary Table S1. Following cycle was used for PCRamplification: 50 °C for 2 min followed by 40 cycles at 95 °Cfor 30 s, 62 °C for 1 min and 72 °C for 20 s. The generationof specific PCR product was confirmed by melting curveanalysis. All samples were run in triplicate. Amplification ofglyceraldehyde-3-phosphate dehydrogenase was used as aninternal control. Basal level of transcript expression in wild

48 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

type (DI700) strain at corresponding time point was used fordata normalization and hence assesses relative abundance.Data analysis was performed using 2−ΔΔCt method.

2.8. Statistical analysis

All proteomic results were analyzed and expressed as mean ±standard deviation (SD). Proteins detected and quantified inonly one biological replicate were not considered for analysis.Non-parametric Kruskal–Wallis test followed by Dunn's testwas used to analyze quantitative real-time PCR (qRT-PCR)data.

3. Results and discussion

3.1. Proteome analysis of DFMO-resistant and wild-typeparasites by iTRAQ

A DFMO-resistant L. donovani strain (DFMO-16) that overpro-duces ornithine decarboxylase has been reported and charac-terized previously [18]. The DFMO-16 strain was isolated bygradually increasing the concentration of DFMO until theparasites were capable of proliferating in 16 mM DFMO. TheDFMO-16 parasites showed an unstable phenotype in that theamplification of the ODC gene, the increased amount of ODCtranscript, the overproduction of ODC activity, and the DFMO-resistance growth phenotype reverted in the absence ofselective pressure [24]. A global approach was utilized tocompare the proteome of DFMO-resistant (DFM0-16) andwild-type L. donovani promastigotes in an effort to identifyproteins that may play a crucial role in DFMO-resistance inLeishmania and to elucidate underlying mechanism causingDFMO resistance.

We divided the output of the comparison between wild-type (DI700) and DFMO-16 strains into three groups. Group I,Group II and Group III represent three individual datasetsobtained from three distinct biological replicate experiments.The Supplementary Table S2 provides all the protein IDs with≥95% confidence and having at least 1 peptide from threeindividual datasets with their respective ratios, pval, andEF-values.

In the case of Group I, a total of 622 proteins were identifiedout of which 487 proteins were having known function andremaining 135 proteins were listed as hypothetical proteins(proteins of unknown function) (Table S2). A total number of429 proteins were identified in Group II with 359 proteinshaving known functions while 70 proteins were hypotheticalproteins (Table S2). Group III contained a total number of 189proteins with 167 proteins of known function while 22 werehypothetical proteins (Table S2). The Supplementary Table S2also includes merged data sets and functional annotations ofall the non-redundant proteins identified in the three groups.A total number of 686 non-redundant proteins were identifiedout of 8184 total theoretical proteins in L. infantum (Table S2). Aconsiderable proportion of hypothetical proteins (167) weredetected out of total 686 non-redundant proteins. The totalnumber of detected proteins represents almost 11.9% of theentire L. infantum proteome (http://www.genedb.org/genedb/linfantum).

Those proteins detected above 95% confidence in theproteomic screen were further functionally classified into10 major functional classes (proteins were assigned theirparticular functional classification based Uniprot, GeneDBand TrytrypDB databases), namely metabolic processes, pro-tein synthesis, stress-related proteins and protein folding,intracellular survival/proteolysis, cell signaling and vesiculartrafficking, protein modification and turnover, cell motilityand cytoskeleton, transport, free-radical detoxification, hypo-thetical and unknown proteins (Fig. 2 and Table S2). Twomajor groups that shared more than half of the total proteinsdetected were metabolic processes (30%) and hypotheticaland unknown proteins (24%) (Fig. 2A). A large number ofproteins (17%) attributed to protein synthesis were detected inthe DFMO-resistant parasites (Fig. 2A). Proteins involved inmetabolic processes were further classified into 10 sub-classes (Fig. 2B).

3.2. Identification and functional annotation of differentiallyabundant proteins in DFMO-resistant parasites

Proteins identified by Protein Pilot software with ≥95%confidence (Unused-ProtScore ≥ 1.3) and iTRAQ ratios ≥1.5 or≤0.5 were considered differentially abundant. Keeping thesefiltering criteria, a list of 101 differentially modulated proteinswere identified (Table 1). Groups I, II and III showed higherabundance of 63, 71 and 49 proteins respectively in theDFMO-16 strain (Table 1, Fig. 3A). Groups I and II had 52proteins that were common to both the groups whereasGroups I and III had 30 common proteins that showed higherabundance in DFMO-16 strain (Table 1, Fig. 3A). We observed38 proteins that were in abundance and common in bothGroups II and III. Out of the 82 proteins that were in abun-dance (Table 1), 19 proteins showed higher levels in all thethree Groups (Table 1, Fig. 3A). Table 1 shows proteins thathad low abundance in DFMO-16 strain. Groups I, II and III hadlower levels of 19, 17 and 12 proteins respectively (Table 1,Fig. 3B). Groups I and II have 17 common proteins that were inlow abundance in DFMO-16 strain. Whereas Groups I and IIIhad 12 proteins that were common to both groups. Groups IIand III had 10 proteins that were in low abundance in bothgroups. A total of 10 proteins were found in low abundanceand were common in all three Groups (Table 1, Fig. 3B).

Marked variation in fold abundance was observed inseveral proteins. This could be due to analysis of threedifferent biological replicates. Variations are bound to occurin protein abundance patterns due to even slight changes intiming and variations in sample handling as well as labelingprocedure. Usually there are less marked variations in tech-nical replicates where a single sample is split into differentreplicates. In the present study, we considered only thoseproteins that had similar fold change in a minimum of twobiological replicates.

Differentially abundant proteins were further separatedinto 18 functional classes. Major functional events that aremodulated and showed enhanced levels in DFMO-resistantparasites include: TCA cycle, nucleoside and nucleotidemetabolism, amino acid and derivative metabolic processes,protein synthesis, stress related proteins, intracellular surviv-al and proteolysis, protein modification and turnover, free

Fig. 2 – Pie-diagram showing relative distribution of Leishmania proteins into 10 major classes depending on their cellularfunction as identified by iTRAQ in Groups I, II and III. Group I, Group II and Group III represent three individual datasetsobtained from three distinct biological replicate experiments. B: Pie diagram depicting distribution of Leishmania proteinsidentified under the class of metabolic processes in to subclass of specific metabolic processes.

49J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

radical detoxification, cell signaling and vesicular trafficking,transport, histones and chromatin remodeling, transcriptionregulation and RNA processing and hypothetical proteins(Fig. 4). Prominent proteins that were in low abundanceincluded proteins involved in the glycolytic pathway, thepentose phosphate shunt and other carbohydrate metabolicprocesses and also proteins involved in countering oxidativeand nitrosative stress (Fig. 4).

3.3. Differential modulation of enzymes involved in polyaminemetabolism

Because DFMO inhibits ODC and thus polyamine biosyn-thesis, proteins involved in this pathway are of obviousinterest in our study. We found an increased abundance ofS-adenosylmethionine synthetase (SAMS) (LinJ.30.3580), cysta-thione β-lyase like protein (LinJ.14.0470), and dihydrofolatereductase (DHFR-TS, LinJ.06.0890).

The enzyme SAMS is essential for the production ofS-adenosylmethionine (AdoMet), a direct precursor for spermi-dine biosynthesis. AdoMet is decarboxylated by S-adenosylme-thionine decarboxylase (AdoMetDC) and in turn provides theaminopropyl moiety for the synthesis of the critical polyamine

spermidine. We observed an increased abundance (~2.5-fold) ofSAMS in the DFMO resistant strain at 24 h and this was validatedat themRNA level using real time quantitative reverse transcrip-tion PCR (qRT-PCR) (Fig. 5A). Higher levels of SAMS and thereaction productAdoMetmayhelp tomaintain spermidine levelsin the DFMO resistant parasites.

Cysteine, besides being crucial in protein biosynthesis andthe synthesis of pivotal biomolecules such as lipoic acid andcoenzymeA, can also contribute toAdoMet formation [25]. Theamino acid is converted to cystathione and homocysteine bythe actions of cystathionine γ-synthase and cystathionineβ-lyase, respectively, and the homocysteine generated is thenrecycled into methionine, the principal source of AdoMet.Enhanced levels of cystathionine β-lyase like protein(LinJ.14.0470) (~1.61-fold) were observed in the DFMO-resistantstrain indicating an increase in methionine biosynthesis tocompensate for the loss of putrescine caused by DFMO-mediated inhibition of ODC. The increased abundance ofcystathionine β-lyase like protein was substantiated usingqRT-PCR (Fig. 5B).

We could not detect ODC in our proteomic screen.However, when we tested the gene expression patterns ofODC (LinJ.12.0100) using qRT-PCR, an increased expression

Table 1 – List of differentially modulated proteins in DFMO-resistant L. donovani (DFMO-16) when compared to the wild-typeL. donovani (DI700).

N Accession#

Name Peptidesa

(95%)% coverage R/W

IR/WII

R/WIII

Meanvalue

SD (±)

Proteins with increased abundanceMetabolic processCarbohydrate metabolic process1 LinJ.10.0310 Isocitrate dehydrogenase [NADP], mitochondrial

precursor, putative14,13,4 42.1,45.3,12.9 1.49b 1.94 1.54 1.65 0.24

2 LinJ.33.2680 Isocitrate dehydrogenase, putative 5,2 20.7,17.4 1.47b 1.45b – 1.46b 0.013 LinJ.26.1530 Trifunctional enzyme alpha subunit,

mitochondrial precursor-like protein1,1 13,8 2.37 – 62.51 32.44 42.52

4 LinJ.29.2080 Fumarate hydratase, putative 4,2 20.8,11.8 1.67 1.76 – 1.71 0.065 LinJ.23.0410 NADP-dependent alcohol dehydrogenase,

putative7, 9, 2 33.8, 47.2, 11.4 1.06c 1.48b 1.54 1.51 0.04

Amino acid and miscellaneous metabolic processes6 LinJ.32.3110 Nucleoside diphosphate kinase b 11,9,2, 80.1,77.5,22.5 1.01c 8.7 1.99 5.34 4.747 LinJ.30.3580 S-adenosylmethionine synthetase 5,3 26.5,23.2 2.37 2.58 – 2.47 0.148 LinJ.14.0470 Cystathionine beta-lyase-like protein 1,1 4.2, 19.6 1.54 1.68 – 1.61 0.099 LinJ.06.0890 Dihydrofolate reductase-thymidylate synthase 2, 2 17.8, 15.8 2.13 – 1.69 1.91 0.3110 LinJ.05.0100 Phosphoprotein phosphatase, putative 6,6,1 30.3,22.5,8.2 1.92 1.12c 12.7 5.24 6.46

Protein metabolismProtein synthesis11 LinJ.36.0210 Elongation factor 2 37, 38, 9 59.2,57.5,19.5 0.99 2.12 1.72 1.61 0.5712 LinJ.17.0110 Elongation factor 1-alpha 53, 26 64.8,51.3 2.63 – 5.15 3.89 1.7813 LinJ.34.0890 Translation elongation factor 1-beta, putative 8,12,4 52.3,60.7,19.3 1.51 1.77 1.49b 1.59 0.1514 LinJ.18.0740 Elongation factor Tu, putative 1,3 11.8,12 2.6 42.85 – 22.7 28.415 LinJ.03.0960 Elongation initiation factor 2 alpha subunit, putative 1,1,1 13.3,12.4,3.9 2.14 1.08c 2.93 2.05 0.9216 LinJ.17.0200 Elongation factor 1-alpha 65,2 75.3, 22.4 – 19.95 14.4 17.17 3.9217 LinJ.28.2480 Eukaryotic translation initiation factor, putative 3,2 12.8, 18.9 – 84.72 3.41 44.06 57.4918 LinJ.36.5870 Isoleucyl-tRNA synthetase, putative 8,11,2 24.4,23.9,9 1.30c 3.28 2.12 2.23 0.9919 LinJ.36.4030 Glycyl tRNA synthetase, putative 3,2 20.4, 17.6 3.9 2.1 – 3.0 1.2720 LinJ.29.2580 60S ribosomal protein L13, putative 10,8,2 62.3,54.1,31.4 2.18 3.07 2.75 2.66 0.4521 LinJ.26.0160 60S ribosomal protein L7, putative 8,6,2 65.1,46,27.8 2.1 2.8 1.45c 2.11 0.6722 LinJ.04.0950 60S ribosomal protein L10, putative 6,4,1 47.9,46,30.1 4.65 4.57 2.65 3.95 1.1323 LinJ.34.2730 Ribosomal protein L3, putative 6,10,3 42.7,37.7,18.1 1.03c 2.35 7.65 3.67 3.524 LinJ.07.0560 60S ribosomal protein L7a, putative 8,6,4 27.3, 23.7, 18.3 2.67 4.74 6.79 5.76 1.4425 LinJ.01.0440 Ribosomal protein S7, putative 6,6 49,28.5 1.72 4.28 – 3 1.8126 LinJ.35.0410 40S ribosomal protein S3A, putative 6, 6, 2 40.2, 44.7, 20.5 1.83 3.9 2.24 2.65 1.0927 LinJ.33.0770 60S ribosomal protein L6, putative 5,4,2 70.3,39,26.2 1.81 2.1 4.78 2.89 1.6328 LinJ.34.3440 60S ribosomal protein L21, putative 6,6,2 54.7,47.2,17 1.03c 5.8 5.34 4.05 2.6329 LinJ.22.1410 40S ribosomal protein L14, putative 4,2,1 27,23.2,14.6 1.1 c 1.73 1.61 1.48 0.3330 LinJ.35.1450 60S ribosomal protein L2, putative 5,3,1 29.2,12.3,8.1 1.94 2.22 1.21c 1.79 0.5231 LinJ.13.1130 40S ribosomal protein S4, putative 4,3,1 34.4,13.1,12.3 3.13 1.94 1.83 2.3 0.7232 LinJ.35.3830 60S ribosomal protein L27A/L29, putative 4,4,3 44.1,52.4,37.2 1.02c 4.44 1.52 2.32 1.8433 LinJ.34.0910 60S ribosomal protein L13a, putative 4,6 35.1,55 1.69 1.92 – 1.8 0.1634 LinJ.36.5350 40S ribosomal protein SA, putative 5,5 40.2,28.9 1.67 2.77 – 2.22 0.7735 LinJ.24.2170 40S ribosomal protein S8, putative 3,2,1 40,26.4,15.9 2.7 3.4 1.55 2.55 0.9336 LinJ.32.0440 60S ribosomal protein L17, putative 2,4,1 33.1,34.9, 6.6 1.87 1.87 1.69 1.81 0.137 LinJ.36.3020 40S ribosomal protein S24e 2,3 44.5,38 2.35 5.39 – 3.87 2.1438 LinJ.35.0600 60S ribosomal protein L18a, putative 2,3,1 22.9,33.5,15.6 1.12c 2.65 3.1 2.29 1.0339 LinJ.11.1130 60S ribosomal protein L28, putative 2,3 36.7,44.2 1.57 3.87 – 2.91 1.6240 LinJ.21.1790 40S ribosomal protein S11, putative 2,3,1 21.3,31.2,5 2.6 12.82 2.60 6.0 5.941 LinJ.29.1920 40S ribosomal protein S15A, putative 2,3 34.6,37.7 2.55 19.23 – 10.89 11.7942 LinJ.35.3340 60S ribosomal subunit protein L31, putative 2,1 31.7,35.5 1.55 2.65 – 2.1 0.7743 LinJ.22.0340 40S ribosomal protein S15, putative 1,1 27.6,24.3 1.62 1.67 – 1.64 0.0344 LinJ.29.2480 60S ribosomal protein L39, putative 1,1,2 54.9,39.2,39.2 2.99 2.14 3.19 2.77 0.5545 LinJ.30.3650 40S ribosomal protein S14 1,4 27.1,22.2 1.99 2.14 – 2.06 0.10646 LinJ.27.2480 60S acidic ribosomal subunit protein 1,1,1 12.1,2.2,10.2 0.99c 87.09 2.07 30.05 49.447 LinJ.36.4730 60S ribosomal protein L18, putative 2,1 14.7,9.6 – 1.85 4.24 3.04 1.68

Stress and protein folding response48 LinJ.28.3060 Heat-shock protein hsp70, putative 82,79,16 67.3,67.1,46.6 1.55 1.48 1.82 1.61 0.1749 LinJ.33.0370 Heat shock protein 83-1 36,42,15 57.3,60.9,30.7 1.69 7.17 1.33c 3.39 3.2750 LinJ.26.1220 Heat shock protein 70-related protein 40,25,8 62.3,51.6,24 2.37 2.22 0.8c 1.79 0.8651 LinJ.33.2520 Heat shock protein, putative 11,11,2 37.2,31.9,11.7 1.81 5.05 1.19c 2.68 2.0752 LinJ.32.1060 Chaperonin containing t-complex protein, putative 5,5,1 23.6,25.8,10.2 2.37 6.6 2.14 3.7 2.51

50 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

Table 1 (continued)

N Accession#

Name Peptidesa

(95%)% coverage R/W

IR/WII

R/WIII

Meanvalue

SD (±)

53 LinJ.27.1150 T-complex protein 1, beta subunit, putative 5,6,1 42.2,26.5,13.4 3.28 1.24c 9.28 4.6 4.1754 LinJ.25.0940 Cyclophilin a 28,25,10 70.6,65.5,41.2 1.43c 2.96 1.54 1.97 0.8555 LinJ.33.1730 Cyclophilin 4, putative 7,6,1 35.3,24,3.1 0.98c 3.8 1.51 2.09 1.49

Intracellular survival and proteolytic virulence factors56 LinJ.14.0920 Calpain-like cysteine peptidase, putative, cysteine

peptidase, Clan CA, family C2, putative8,13,1 79.1,99.1,20 2.35 13.55 0.77c 5.55 6.96

57 LinJ.35.2400 Aminopeptidase P, putative, metallo-peptidase,Clan MG, Family M24

1,1 8.9,7.2 43.6 1.87 – 22.7 29.5

58 LinJ.23.1120 Cytosolic leucyl aminopeptidase,metallo-peptidase, clan MF, family M17

1, 1 19.4, 13.8 1.87 – 1.9 1.88 0.02

59 LinJ.02.0680 ATP-dependent Clp protease subunit, heat shockprotein 78 (HSP78), putative, serine peptidase,putative

6,4,2 36.6,19.8,12.7 2.51 1.49b 1.52 1.84 0.58

Protein turnover and modification60 LinJ.09.0950 Polyubiquitin 10,13 67.3,77 1.7 3.43 – 2.56 1.2261 LinJ.29.0810 U-box domain protein, putative 2,2,1 11.8,7.5,3.8 1.95 3.25 4.69 3.29 1.37

Free radical detoxification62 LinJ.29.1250 Tryparedoxin 11,10 62.1,51 2.37 1.73 – 2.05 0.4563 LinJ.29.1240 tryparedoxin 5,2 48.3,33.6 2.16 1.81 – 1.98 0.24

Cell signaling and vesicular trafficking64 LinJ.24.1570 IgE-dependent histamine-releasing factor,

putative12,14,2 55.3,68.8,14.7 2.03 5.44 5.15 4.2 1.89

65 LinJ.29.0950 ADP ribosylation factor 3, putative 4,6 53.6,50.8 3.16 1.6 – 2.39 1.166 LinJ.36.3360 14-3-3 protein-like protein 3,2 41.5,15.5 2.2 1.62 – 1.91 0.4167 LinJ.35.1020 Casein kinase I, putative 1,1 8,13.5 1.51 1.75 – 1.63 0.1668 LinJ.21.1490 Adenylate kinase, putative 1,1 16.7,8.9 1.75 2.58 – 2.16 0.58

Transport69 LinJ.19.0200 ADP, ATP carrier protein 1, mitochondrial precur-

sor, putative, ADP/ATP translocase 1, putative3,4,2 23,24,12.3 2.67 1.33 4.13 2.71 1.4

70 LinJ.29.0640 ATP-binding cassette protein subfamily A, mem-ber 10, putative, ABC transporter, putative

2,1 7,2.8 1.83 – 1.51 1.67 0.22

71 LinJ.14.1050 ADP/ATP mitochondrial carrier-like protein 1,1 4.8, 14.6 – 72.4 1.7 37.05 49.972 LinJ.35.4490 Mitochondrial phosphate transporter, putative 1,1 15.4, 16.7 1.75 1.58 – 1.66 0.12

Histones and chromatin remodeling73 LinJ.21.1170 Histone H2A, putative 2,4 34.9,31.1 2.31 3.16 – 2.73 0.674 LinJ.10.1070 Histone H3 2,4 24.6,33.1 2.8 5.29 – 4.04 1.7675 LinJ.06.0010 Histone H4 3, 2 61,41.2 – 3.45 3.49 3.47 0.02

Transcription regulation and RNA processing76 LinJ.32.0410 ATP-dependent RNA helicase, putative 11,9,1 45.8,36.6,5.9 1.67 5.54 5.86 5.7 0.2277 LinJ.23.0930 Mitochondrial RNA binding protein, putative 9,6 47.7,35.6 1.61 3.94 – 2.77 1.6478 LinJ.35.0370 ATP-dependent DEAD-box RNA helicase, putative 3,2,2 19.5,12.6,8.6 1.81 1.5 1.24c 1.51 0.28

Cell motility and cytoskeleton79 LinJ.04.1250 Actin 6,7,1 34.6,35.6,13.6 1.02c 2.16 2.03 1.73 0.62

Hypothetical proteins80 LinJ.14.0190 Hypothetical protein, conserved 7,6,1 59.3,31.2,14.1 0.96c 1.48b 2.16 1.53 0.681 LinJ.35.4540 Hypothetical protein, conserved 3,5,1 22.1,35.7,17.6 1.78 1.26b 1.51 1.51 0.2682 LinJ.25.0550 Hypothetical protein SCD6.10 2,1,1 23.9,13.1,9.8 3.31 1.35c 7.44 4.02 3.1

Proteins with decreased abundanceMetabolic processesCarbohydrate metabolism1 LinJ.36.1320 Fructose-1,6-bisphosphate aldolase 15,14,1 61.7,44.7,9.7 0.17 0.13 0.18 0.16 0.022 LinJ.36.3630 2-Oxoglutarate dehydrogenase E1 component,

putative2,2 19.4,11.9 0.44 0.55b – 0.49 0.07

3 LinJ.28.2600 2-Oxoglutarate dehydrogenase, E2 component,dihydrolipoamide succinyltransferase, putative

4,6,1 22.9,18.8,16.5 0.5 0.29 0.2 0.33 0.15

4 LinJ.34.0080 Glucose-6-phosphate 1-dehydrogenase, putative 7,4,3 32.6,24,11.7 0.41 0.64c 0.4 0.48 0.135 LinJ.27.2500 Glycosomal phosphoenolpyruvate carboxykinase,

putative14,15,2 44.4,36.6,18.9 0.44 0.39 0.45 0.42 0.03

6 LinJ.35.0990 Aldose 1-epimerase, putative 4,4,1 20,26.4,17.5 0.46 0.52b 0.25 0.41 0.147 LinJ.11.1000 Pyruvate phosphate dikinase, putative 14,15,4 48,34.5,17.2 0.49 0.44 0.52b 0.48 0.048 LinJ.23.0860 3-Ketoacyl-CoA thiolase, putative 10,9,6 39,41.3,32.2 0.52 0.44 0.22 0.39 0.15

(continued on next page)

Protein metabolismStress and protein folding response

51J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

Table 1 (continued)

N Accession#

Name Peptidesa

(95%)% coverage R/W

IR/WII

R/WIII

Meanvalue

SD (±)

9 LinJ.16.1380 Cytochrome c, putative 6,5,1 35.9,43.6,25 0.44 0.3 0.33 0.35 0.0710 LinJ.03.0190 Delta-1-pyrroline-5-carboxylate dehydrogenase,

putative18,14 51.8,43.2 0.48 0.48 – 0.48 0.0

Protein metabolismProtein synthesis11 LinJ.03.0410 60S acidic ribosomal protein P2, putative 1,1 26.8,33.9 0.42 0.27 – 0.34 0.106

Stress and protein folding response12 LinJ.28.1310 Glucose-regulated protein 78, putative 24,19,4 50.3,59.7,21.3 0.42 0.27 0.8c 0.49 0.2713 LinJ.31.2670 Calreticulin, putative 8,8,2 23,20.3,14 0.27 0.19 0.38 0.28 0.0914 LinJ.06.1090 Protein disulfide isomerase 4,1,1 39.1,14.3,11.3 0.54b 0.64c 0.27 0.48 0.19

Intracellular survival and proteolytic virulence factors15 LinJ.20.1210 Calpain-like cysteine peptidase, putative, cysteine

peptidase, Clan CA, family C2, putative4,7,2 17.5,22.5,6.1 0.51b 0.4 0.52b 0.47 0.06

Protein turnover and modification16 LinJ.12.0003 Proteasome beta-1 subunit, putative 2,2 12,24 0.39 0.42 – 0.4 0.02

Cell motility and cytoskeleton17 LinJ.05.0380 Microtubule-associated protein, putative 20,11,2 83.5,87.5,75.2 0.34 0.17 0.55b 0.35 0.19

Hypothetical and uncharacterized proteins18 LinJ.02.0520 Hypothetical protein, unknown function 5,2 40.5,28.2 0.42 0.3 – 0.36 0.0819 LinJ.15.0320 Ribonucleoprotein p18, mitochondrial precursor,

putative6,4 62,44.9 0.45 0.47 – 0.46 0.01

The table contains quantitative information of the proteins from DFMO-resistant L. donovani (DFMO16) compared with its wild type parentalcontrol (DI700). These proteins fulfill the criteria (i.e. unused ProtScore ≥ 1.3, FDR = 5%, change in abundance levels of at least ≥1.5 (increasedabundance) or at least ≤0.5 (decreased abundance) as defined in the experimental procedures).R: DFMO-16; W: DI700; R/W: Fold abundance ratio.SD (±): Standard deviation.a The total number of identified peptides with ≥95% confidence.b Values very near to filtering criteria of fold abundance.c Values away from filtering criteria of fold abundance.

Metabolic processesCarbohydrate metabolism

Fig. 3 – Venn diagram showing proteins identified using isobaric tagging for relative and absolute quantification(iTRAQ/LC–MS/MS). Three biological replicates are indicated as Groups I, II and III respectively. A: Proteins with increasedabundance. B: Proteins with decreased abundance.

52 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

Fig. 4 – Bar diagram showing the percentage of increased and decreased abundance of proteins in DFMO-resistant Leishmaniadonovani based on their functional classification.

53J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

(>25-fold) was observed in DFMO-resistant parasites at 12 htime point as compared to its wild-type parental counterpart(DI700). At later time points (24 and 48 h) ODC showed adecrease in the expression levels (Fig. 5F).

Furthermore, an augmented abundance of DHFR-TS(LinJ.06.0890) (~1.91-fold) was observed in DFMO-resistantparasites. Dihydrofolate reductase (DHFR) occurs as a bifunc-tional enzyme in Leishmania, joined to thymidylate synthase(DHFR-TS), an NADPH-dependent enzyme required to gener-ate H4-Folate [26]. Tetrahydrofolates can be further utilized inmethionine and NADPH synthesis, where methionine can beused for spermidine synthesis and NADPH as a reducingequivalent for the trypanothione system.

3.4. Key proteins modulating carbohydrate and energymetabolism

Free radical detoxification depends on reducing equivalentsand key intermediates involved in T[SH]2 biogenesis [27]. Wefound increased levels of several proteins in the DFMO-16strain that are involved in the TCA cycle and fatty acidmetabolism and may provide reducing equivalents needed tosustain thiol-based antioxidant system. Two putative isocitratedehydrogenases (LinJ.33.2680 and LinJ.10.0310) exhibited en-hanced levels, ~1.65-fold and 1.46-fold, respectively, in theDFMO resistant strain. Isocitrate dehydrogenase is known toconvert isocitrate to oxoglutarate and NADPH is formed as abyproduct. In addition, the trifunctional enzyme alpha subunit,

mitochondrial precursor-like protein (LinJ.26.1530) showed sig-nificant increase (32.4-fold) in comparison to the parental DI700strain. As part of the mitochondrial trifunctional enzymecomplex, also known as 3-ketoacyl-CoA thiolase, the enzymeis involved in β-oxidation of fatty acid, during which coenzymeA and NADPH is formed. Furthermore, a putative fumaratehydratase (LinJ.29.2080) (~1.7-fold) and a putative NADP-dependent alcohol dehydrogenase (LinJ.23.0410) (1.5-fold),both enzymes are source of key metabolic intermediates andNADPH, were also found in increased abundance.

We found low abundance of two components of theoxoglutarate dehydrogenase (or α-ketoglutarate dehydroge-nase) complex. The putative 2-oxoglutarate dehydrogenase E1component (LinJ.36.3630), and 2-oxoglutarate dehydrogenase,E2 component, dihydrolipoamidesuccinyltransferase, putative(LinJ.28.2600) were observed at ~0.5-fold and ~0.33-fold lowerlevels respectively, in the resistant strain. This oxoglutaratedehydrogenase multienzyme complex is required to convertα-ketoglutarate to succinyl CoA and a downregulation of thiscomplexmay be a favorablemechanism in DFMO-resistance asα-ketoglutarate is a key constituent in TCA cycle and a valuableintermediate in several of amino acid biosynthesis pathways.

Several glycosomal enzymes like fructose-1,6-bisphosphatealdolase (LinJ.36.1320), glucose-6-phosphate 1-dehydrogenase,putative (LinJ.34.0080) showed reduced abundance in the DFMOresistant strain. Glycosomal phosphoenolcarboxykinase, puta-tive (LinJ.27.2500) that catalyzes the formation of phosphoenol-pyruvate from oxaloacetate also showed reduced abundance

Fig. 5 – Quantitative real-time PCR analysis of gene expression in α-difluoromethylornithine (DFMO) resistant L. donovani (DFMO16) as compared to its wild-type parentalcontrol (DI700). Leishmania glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Basal level of transcript expression in wild type (DI700) strainat corresponding time point was used for data normalization and hence assesses relative expression. Data analysis was performed using 2−ΔΔCt method. Scatter plot showsthree technical replicates with similar results and error bars represent standard error mean. Horizontal bars indicate median values, and the p values shown are from thenon-parametric Kruskal–Wallis test followed by Dunn's test. *p < 0.05.

54JO

UR

NA

LO

FPR

OT

EO

MIC

S102

(2014)

44–59

55J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

thus may alternatively increase the pool of oxaloacetate toundergo amino acid biosynthesis.

3.5. Proteins involved in free radical detoxification

Kinetoplastids enjoy a unique peroxide detoxification whichencompasses trypanothione (T [SH]2), trypanothione reduc-tase (TR), the thioredoxin related protein tryparedoxin (TXN/TPN), 2-Cys-peroxiredoxin-type, and tryparedoxin peroxidase(TPX) [28]. The T[SH]2/TR axis is the fundamental source ofdetoxification of oxidative and nitrosative stresses duringparasite infection and multiplication inside the parasito-phorous vacuole [29]. Peroxidases that detoxify hydroperoxideand peroxynitrite (ONOO−) are driven by reducing equivalentsderived from trypanothione (T[SH]2) which itself is keptreduced by NADPH-dependent flavoenzyme trypanothionereductase (TR). We found an increased abundance (~2.0-fold)of tryparedoxin (LinJ.29.1250 and LinJ.29.1240) in the DFMOresistant strain. Tryparedoxins not only react with differentperoxidases but also with other substrates like ribonucleotidereductase and Universal Minicircle Sequence Binding Protein(UMSBP) at the kinetoplast DNA replication origin [30]. Thus,tryparedoxin not only aids in hydroperoxide metabolism butalso plays a role in deoxynucleotide synthesis and mitochon-drial DNA replication essential for parasite proliferation. Al-though we could detect the presence of key enzymes liketryparedoxin peroxidase (LinJ.15.1100 and LinJ.15.1120), a pu-tative Fe-SOD (LinJ.32.1910 and LinJ.08.0300) and peroxidoxin(LinJ.23.0050) involved in trypanothione based free radicaldetoxification process, these proteins were not identified asmodulated in the proteomic screen. The pattern of geneexpression of these enzymes was then tested using real-timePCR in DFMO-resistant promastigotes as compared to thewild-type (DI700) strain. Interestingly, a higher level of geneexpression of peroxidoxin (LinJ.23.0050), tryparedoxin perox-idase (LinJ.15.1100) and Fe-SOD, putative (LinJ.32.1920), wasdetected at 24 h in the DFMO-resistant promastigotes (Fig. 5C,D and F). Tryparedoxin peroxidases employ TXN as a source ofreducing electrons during peroxide exclusion and they includemembers of peroxidoxin and non-selenium glutathione perox-idase families. These results show a dynamic and quantitativealteration in the expression of key cellular enzymes, mostlyperoxidases, involved in trypanothione mediated thwarting ofperoxides and peroxynitrates in DFMO-resistant parasites.

3.6. Increased abundance of parasite proteases inDFMO-resistant parasites

Protozoan proteases play crucial roles in life cycle transition,host cell invasion, migration through tissue barriers, immuneevasion and activation of inflammation in the mammalianhost [31]. We observed a general trend of increased abundanceof these parasite proteases in DFMO resistant parasites.Cysteine-proteases (CPs) are characterized as prominent viru-lence factors in Leishmania mexicana [31]. Cysteine-proteasesCPA, CPB and CPC, all of which are papain like and belong tothe same group of CPs, clan CA that is divided into differentfamilies. They appear to be isoforms. Calpain-like cysteinepeptidase (LinJ.14.0920) was more abundant in the DFMO-resistant parasites as compared to the parental strain.

However, another isoform, calpain-like cysteine peptidaseputative, clan CA, family C2 putative (LinJ. 20.1210) was foundto be less abundant.

Calpain-like cysteine peptidase (LinJ.14.0920) (~5.5-fold),metallo-peptidases like cytosolic leucyl aminopeptidase,metallopeptidase (LinJ.23.1120) (1.9-fold) and aminopep-tidase P (LinJ.35.2400) were found in increased abundance(~23.0-fold) in the resistant strain. An increased abundance ofa putative ATP-dependent Clp protease subunit (LinJ.02.0680),a third class of proteases (serine proteinases), was alsoobserved. This class of protease plays an important rolein infection and its maintenance [32]. These results suggestan increased level of parasite proteases as a key mecha-nism operating in DFMO-resistant Leishmania parasites,which may lead to an effective immune suppression inthe host.

3.7. Stress and protein folding response

Heat shock proteins are highly conserved proteins that evolvedto counteract a wide range of physiological and environmentalinsults including heat, reactive oxygen species and anticancerdrugs [33,34].We foundan increased levels of several heat shockproteins in the DFMO resistant strain, including a putativeheat-shock protein hsp70 (LinJ.28.3060) (~1.6-fold), HSP70-related protein (LinJ.26.1220) (~1.8-fold), HSP 83-1 (LinJ.33.0370)(~3.4-fold) and another putative heat shock protein (LinJ.33.2520)(~2.7 fold). Heat shock proteins probably counteract DFMO-induced drug pressure in Leishmania parasites to increase itssurvival. Cyclophilin a (LinJ.25.0940) and a putative cyclophilin 4(LinJ.33.1730) were also found in significantly increased abun-dance (~2.0-fold and ~2.1-fold respectively) in the DFMOresistant strain. Cyclophilins play an important role as ER-resident chaperons during oxidative stress [35]. Together withparasitic proteases and heat shock proteins, cyclophilin mayalso play an important role in parasite survival and multiplica-tion in DFMO resistant parasites.

3.8. Proteins involved in signal transduction

Subversion of signaling networks is a classicalway to reprograma delicate balance of homeostasis inside a biological system [36].We found an increased abundance of two proteins that areinvolved in signal transduction in the DFMO resistant strain: aputative ADP ribosylation factor 3 (LinJ.29.095) (~2.4-fold) and14-3-3 protein-like protein (LinJ.36.3360) (~1.9-fold). BecauseL. donovani ADP ribosylation factor 1 (LdARF1) has been charac-terized and plays a crucial role in Golgi-mediated vesiculartrafficking [37], it is feasible that the putative ADP ribosylationfactor 3 is also involved in trans-Golgi networks. Mammalian14-3-3 proteins act as adapter in signaling cascades and play animportant role in small GTPase mediated signal transductionand thus vesicular trafficking [38,39]. It appears that GTPasesignaling may be crucially reprogrammed in DFMO resistantparasites to increase vesicular trafficking.

Furthermore, we observed two protein kinases, a putativecasein kinase I (LinJ.35.1020) and a putative adenylate kinase(LinJ.21.1490) at higher abundance (~1.6-fold and ~2.1-foldrespectively). Casein kinase I belongs to the protein kinasesuperfamily and plays a critical role in protein serine/threonine

56 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

kinase activity and helps in parasite survival and adaptationto environment [40]. Adenylate kinases in Leishmania help tomaintain constant intracellular level of adenine nucleotidesnecessary for energy metabolism and nucleic acid biosynthesis[41,42].

3.9. Mitochondrial transporter proteins are upregulated inDFMO-resistant parasites

The mitochondrial ADP/ATP carrier (AAC) belongs to themitochondrial carrier family (MCF) that plays a crucial role inregulation of energy metabolism as it acts as a key energeticlink between cytosol and mitochondria as it provides ADPand ATP, the substrates/products of mitochondrial ATP syn-thase [43,44]. We found two such putative translocators, aputative ADP, ATP carrier protein 1 (mitochondrial precursor)(LinJ.19.0200) and ADP/ATP mitochondrial carrier-like protein(LinJ.14.1050) in increased abundance (~2.7-fold and 37.0-foldrespectively) in DFMO-resistant parasites. A mitochondrialphosphate transporter (LinJ.35.4490) was also found in in-creased abundance (1.66-fold) in this study. In addition, wefound a putative ATP-binding cassette protein subfamily A,member 10 (LinJ.29.0640), at higher levels (~1.67-fold) inDFMO-resistant strain, thus compounding the role of theMCF family and ABC transporters in maintaining exchange ofreaction intermediates like ADP, ATP and nucleotides underdifferent physiological conditions like prolonged drug abusesand stage differentiation in parasites [45].

3.10. Effect on protein synthesis and ribosomal proteins

Anticancer drugs have been known to affect key regulators ofthe protein synthetic machinery and deregulation of transla-tion is one of the key features in drug resistant cancer cells[46]. In this study, we detected an increased abundance ofa large number of ribosomal proteins as well as severaleukaryotic initiation and elongation factors. Polyamines likespermidine are known to stimulate the rate of proteinsynthesis and contribute to the specificity of codon/anticodoninteractions and thereby increase the fidelity of proteinsynthesis [47]. We detected a higher abundance of a putativeisoleucyl-tRNA synthetase (LinJ.36.5870) (~2.2-fold) and aputative glycyl–tRNA synthetase (LinJ.36.4030) (~3.0-fold) inthe DFMO-resistant parasites.

3.11. Proteins involved in gene regulation

We observed a general trend of increased abundance amonghistoneproteins. AputativehistoneH2A (LinJ.21.1170) (~2.7-fold),histone H3 (LinJ.10.1070) (~4.0-fold), and histone H4 (LinJ.06.0010)(3.5-fold) were found at distinctly higher levels in the resistantparasites, further suggesting their role in transcriptional changesresulting in increased protein synthesis.

3.12. Other proteins

A large number of hypothetical proteins having unknownfunctions were discovered in either increased or decreasedabundance and future studies of these proteins and their hiddenrole for maintaining drug resistance in parasites is warranted.

4. Conclusions

Mass spectrometry based strategies for quantitative proteo-mics currently plays a crucial role in investigating thephysiological adaptation mechanisms in drug resistant para-sites and hence identification of critical biosignatures thatplay an important role in the mechanism of drug resistanceand its link to drug treatment outcome. The main objective ofthe present study was to obtain a snapshot of the globalevents in order to gain insight into the adaptive responses inLeishmania resistant to DL-α-difluoromethylornithine (DFMO).Relative and absolute quantification of proteins using iTRAQanalysis were applied to compare the proteome of a DFMO-resistant laboratory strain of L. donovani (DFMO-16) with thatof its wild-type parental clone.

As a result, 101 proteins were identified with significantalterations in abundance with 82 proteins found at increasedabundance while 19 proteins were less abundant in the DFMO-resistant parasites (Table 1). Marked variation in fold abun-dance in several proteins was observed that can be due toseparate biological replicates used in the study.

DFMO-induced drug resistance in L. donovani is a complexnetwork of events (Table 1) where parasites show a dynamicmodulation in critical enzymes involved in polyamine bio-synthesis. We propose a model (Fig. 6) for these molecularevents. Several biochemical events were reprogrammed inα-difluoromethylornithine resistant L. donovani having thepotential to increase the pathogen's capacity to survive ahigher drug concentration. This acquired resistance againstDFMO is an adaptive response to defend against drug inducedpolyamine and trypanothione depletion and hence oxidativestress. Several enzymes involved in polyamine precursor syn-thesis, namely SAMS (LinJ.30.3580) and cystathione β-lyase likeprotein (LinJ.14.0470) were present at higher levels in theresistant parasites. S-adenosylmethionine is a key metaboliteand important precursor in the biosynthesis of polyamines likespermidine, which is also necessary for the production of T[SH]2.Increased levels of SAMScanbepostulated to aid adrug resistantphenotype by promoting augmented level of intracellular poolsof spermidine andT[SH]2. SAMalso acts as amethyl group donorin methylation reactions modifying DNA, RNA and histonesthat regulate cell signaling, gene expression and metabolicpathways [48]. Given the fact that the control of gene expressionin Leishmania is largely regulated at post-transcriptional leveland environment factors provide important cues for itsadaptive and evasive responses against hostile environmentinside host and against drug abuses, SAM can be considered tobe an important hub molecule, possibly bridging a cross-talkbetween metabolic and resistant phenotype. Parasites likeLeishmania and trypanosomatids use trypanothione (T [SH]2)as the principal reductant to maintain the redox balance.Trypanothione ismaintained in its reduced state at the expenseof NADPHmediated by trypanothione reductase (TR), membersof the large and well-characterized family of FAD-dependentNAD(P)H oxidoreductases [3]; thus the T[SH]2/TR system inparasites is essentially regulated by NADPH and polyamines(precursors of T[SH]2 biosynthesis). Enzymes in TCA cycleshowed enhancement in DFMO-resistant parasites providingcrucial reaction intermediates in amino acid biosynthesis and

Fig. 6 – Model depicting metabolic reprogramming in DFMO-resistant L. donovani. Red arrow represents altered proteinabundance.

57J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

NADPH needed to fuel T[SH]2 based free radical detoxificationsystem.

This study gives an insight into dynamically alteredphysiological events and identifies key biomarkers in DFMO-resistant parasites providing a deeper understanding of thephenomenon of drug-resistance. Such knowledge is neces-sary to safeguard the efficacy of the currently available pool ofdrugs against leishmaniasis.

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jprot.2014.02.030.

Acknowledgments

Alok K Singh is a recipient of funding from the Council forScientific and Industrial Research, New Delhi, India. Rentala

58 J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

Madhubala is a JC Bose National Fellow. The work is supportedby a grant from theCouncil of Scientific and Industrial Research,India and DST-PURSE grant to RMB. We would like to ac-knowledge the usage of the Advance Instrumentation ResearchFacility (AIRF) at the Jawaharlal University.

R E F E R E N C E S

[1] Ouellette M, Drummelsmith J, Papadopoulou B. Leishmaniasis:drugs in the clinic, resistance and new developments. DrugResist Updat 2004;7:257–66.

[2] Sundar S, More DK, SinghMK, Singh VP, Sharma S,Makharia A,et al. Failure of pentavalent antimony in visceral leishmaniasisin India: report from the center of the Indian epidemic. ClinInfect Dis 2000;31:1104–7.

[3] Colotti G, Ilari A. Polyamine metabolism in Leishmania:from arginine to trypanothione. Amino Acids2011;40:269–85.

[4] Roberts SC, Jiang Y, Gasteier J, Frydman B, Marton LJ, Heby O,et al. Leishmania donovani polyamine biosynthetic enzymeoverproducers as tools to investigate the mode of action ofcytotoxic polyamine analogs. Antimicrob Agents Chemother2007;51:438–45.

[5] Manta B, Comini M, Medeiros A, Hugo M, Trujillo M, Radi R.Trypanothione: a unique bis-glutathionyl derivative intrypanosomatids. Biochim Biophys Acta Gen Subj2013;1830:3199–216.

[6] Fairlamb AH, Blackburn P, Ulrich P, Chait BT, Cerami A.Trypanothione: a novel bis(glutathionyl)spermidine cofactorfor glutathione reductase in trypanosomatids. Science1985;227:1485–7.

[7] Persson L. Ornithine decarboxylase and S-adenosylmethioninedecarboxylase in trypanosomatids. Biochem Soc Trans2007;35:314–7.

[8] Boitz JM, Yates PA, Kline C, Gaur U, Wilson ME, Ullman B, et al.Leishmania donovani ornithine decarboxylase is indispensablefor parasite survival in the mammalian host. Infect Immun2009;77:756–63.

[9] Li F, Hua SB, Wang CC, Gottesdiener KM. Trypanosoma bruceibrucei: characterization of an ODC null bloodstream formmutant and the action of alpha-difluoromethylornithine. ExpParasitol 1998;88:255–7.

[10] Gilroy C, Olenyik T, Roberts SC, Ullman B. Spermidinesynthase is required for virulence of Leishmania donovani.Infect Immun 2011;79:2764–9.

[11] Roberts SC, Tancer MJ, Polinsky MR, Gibson KM, Heby O,Ullman B. Arginase plays a pivotal role in polyamineprecursor metabolism in Leishmania. Characterization of genedeletion mutants. J Biol Chem 2004;279:23668–78.

[12] Carrillo C, Cejas S, Cortés M, Ceriani C, Huber A, González NS,et al. Sensitivity of trypanosomatid protozoa to DFMO andmetabolic turnover of ornithine decarboxylase. BiochemBiophys Res Commun 2000;279:663–8.

[13] Bacchi CJ, Nathan HC, Hutner SH, McCann PP, Sjoerdsma A.Polyamine metabolism: a potential therapeutic target intrypanosomes. Science 1980;210:332–4.

[14] Van Nieuwenhove S, Schechter PJ, Declercq J, Boné G, Burke J,Sjoerdsma A. Treatment of gambiense sleeping sickness in theSudan with oral DFMO (DL-alpha-difluoromethylornithine), aninhibitor of ornithine decarboxylase; first field trial. Trans R SocTrop Med Hyg 1985;79:692–8.

[15] Kaur K, Emmett K, McCann PP, Sjoerdsma A, Ullman B. Effectsof DL-alpha-difluoromethylornithine on Leishmania donovanipromastigotes. J Protozool 1986;33:518–21.

[16] Reguera RM, Fouce RB, Cubría JC, Bujidos ML, Ordóñez D.Fluorinated analogues of L-ornithine are powerful inhibitors

of ornithine decarboxylase and cell growth of Leishmaniainfantum promastigotes. Life Sci 1995;56:223–30.

[17] Heby O, Persson L, Rentala M. Targeting the polyaminebiosynthetic enzymes: a promising approach to therapy ofAfrican sleeping sickness, Chagas' disease, and leishmaniasis.Amino Acids 2007;33:359–66.

[18] Coons T, Hanson S, Bitonti AJ, McCann PP, Ullman B.Alpha-difluoromethylornithine resistance in Leishmaniadonovani is associated with increased ornithine decarboxylaseactivity. Mol Biochem Parasitol 1990;39:77–89.

[19] Bacchi CJ, Garofalo J, Ciminelli M, Rattendi D, Goldberg B,McCann PP, et al. Resistance to DL-alpha-difluoromethylornithine by clinical isolates of Trypanosomabrucei rhodesiense. Role of S-adenosylmethionine. BiochemPharmacol 1993;46:471–81.

[20] Iovannisci DM, Ullman B. High efficiency plating method forLeishmania promastigotes in semidefined orcompletely-defined medium. J Parasitol 1983;69:633–6.

[21] Kruger NJ. The Bradford method for protein quantitation.Methods Mol Biol 1994;32:9–15.

[22] Guo Y, Singleton PA, Rowshan A, Gucek M, Cole RN, GrahamDRM, et al. Quantitative proteomics analysis of humanendothelial cell membrane rafts evidence of MARCKS andMRP regulation in the sphingosine 1-phosphate-inducedbarrier enhancement. Mol Cell Proteomics 2007;6:689–96.

[23] Biyani N, Singh AK, Mandal S, Chawla B, Madhubala R.Differential expression of proteins in antimony-susceptibleand -resistant isolates of Leishmania donovani. Mol BiochemParasitol 2011;179:91–9.

[24] Hanson S, Adelman J, Ullman B. Amplification and molecularcloning of the ornithine decarboxylase gene of Leishmaniadonovani. J Biol Chem 1992;267:2350–9.

[25] Nozaki T, Ali V, Tokoro M. Sulfur-containing amino acidmetabolism in parasitic protozoa. In: Baker JR, RM, DR, editors.Advances in parasitology, vol. 60. Academic Press; 2005. p. 1–99.

[26] Cunningham ML, Beverley SM. Pteridine salvage throughoutthe Leishmania infectious cycle: implications for antifolatechemotherapy. Mol Biochem Parasitol 2001;113:199–213.

[27] Irigoín F, Cibils L, Comini MA, Wilkinson SR, Flohé L, Radi R.Insights into the redox biology of Trypanosoma cruzi:trypanothione metabolism and oxidant detoxification. FreeRadic Biol Med 2008;45:733–42.

[28] Krauth-Siegel RL, Comini MA. Redox control intrypanosomatids, parasitic protozoawith trypanothione-basedthiol metabolism. Biochim Biophys Acta Gen Subj2008;1780:1236–48.

[29] Dumas C, Ouellette M, Tovar J, CunninghamML, Fairlamb AH,Tamar S, et al. Disruption of the trypanothione reductasegene of Leishmania decreases its ability to survive oxidativestress in macrophages. EMBO J 1997;16:2590–8.

[30] Castro H, Tomás AM. Peroxidases of trypanosomatids.Antioxid Redox Signal 2008;10:1593–606.

[31] Silva-Almeida M, Pereira BAS, Ribeiro-Guimarães ML, AlvesCR. Proteinases as virulence factors in Leishmania spp.infection in mammals. Parasit Vectors 2012;5:160.

[32] Contreras I, Gómez MA, Nguyen O, Shio MT, McMaster RW,Olivier M. Leishmania-induced inactivation of themacrophage transcription factor AP-1 is mediated by theparasite metalloprotease GP63. PLoS Pathog 2010;6:e1001148.

[33] Li GC. Heat shock proteins: role in thermotolerance, drugresistance, and relationship to DNA topoisomerases. NCIMonogr 1987:99–103.

[34] Murphy ME. The HSP70 family and cancer. Carcinogenesis2013;34(16):1181–8.

[35] Yurchenko V, Xue Z, Sherry B, Bukrinsky M. Functionalanalysis of Leishmania major cyclophilin. Int J Parasitol2008;38:633–9.

[36] Olivier M, Gregory DJ, Forget G. Subversion mechanisms bywhich Leishmania parasites can escape the host immune

59J O U R N A L O F P R O T E O M I C S 1 0 2 ( 2 0 1 4 ) 4 4 – 5 9

response: a signaling point of view. Clin Microbiol Rev2005;18:293–305.

[37] Porter-Kelley JM, Gerald NJ, Engel JC, Ghedin E, Dwyer DM.LdARF1 in trafficking and structural maintenance of thetrans-Golgi cisternal network in the protozoan pathogenLeishmania donovani. Traffic 2004;5:868–83.

[38] Darling DL, Yingling J, Wynshaw‐Boris A. Role of 14-3-3proteins in eukaryotic signaling and development. In:Schatten Gerald P, editor. Current topics in developmentalbiology, vol. 68. Academic Press; 2005. p. 281–315.

[39] Gelperin D, Weigle J, Nelson K, Roseboom P, Irie K,Matsumoto K, et al. 14-3-3 proteins: potential roles invesicular transport and Ras signaling in Saccharomycescerevisiae. Proc Natl Acad Sci U S A 1995;92:11539–43.

[40] Allocco JJ, Donald R, Zhong T, Lee A, Tang YS, HendricksonRC, et al. Inhibitors of casein kinase 1 block the growth ofLeishmania major promastigotes in vitro. Int J Parasitol2006;36:1249–59.

[41] YanH, TsaiMD. Nucleosidemonophosphate kinases: structure,mechanism, and substrate specificity. Adv Enzymol Relat AreasMol Biol 1999;73:103–34 [x].

[42] Villa H, Pérez-Pertejo Y, García-Estrada C, Reguera RM,Requena JM, Tekwani BL, et al. Molecular and functional

characterization of adenylate kinase 2 gene from Leishmaniadonovani. Eur J Biochem 2003;270:4339–47.

[43] Traba J, Satrústegui J, del Arco A. Adenine nucleotidetransporters in organelles: novel genes and functions. CellMol Life Sci 2011;68:1183–206.

[44] Chan KW, Slotboom D-J, Cox S, Embley TM, Fabre O, van derGiezen M, et al. A novel ADP/ATP transporter in themitosomeof the microaerophilic human parasite Entamoeba histolytica.Curr Biol 2005;15:737–42.

[45] Klingenberg M. The ADP and ATP transport in mitochondriaand its carrier. Biochim Biophys Acta Biomembr2008;1778:1978–2021.

[46] Fu Z, Fenselau C. Proteomic evidence for roles for nucleolinandpoly[ADP-ribosyl] transferase in drug resistance. J ProteomeRes 2005;4:1583–91.

[47] Nishimura K, Okudaira H, Ochiai E, Higashi K, Kaneko M, IshiiI, et al. Identification of proteins whose synthesis ispreferentially enhanced by polyamines at the level oftranslation in mammalian cells. Int J Biochem Cell Biol2009;41:2251–61.

[48] Loenen WAM. S-adenosylmethionine: jack of all trades andmaster of everything? Biochem Soc Trans 2006;34:330–3.