Up-regulation of silent information regulator 2 (Sir2) is associated with amphotericin B resistance in clinical isolates of Leishmania donovani

Up-regulation of silent information regulator 2 (Sir2) is associated withamphotericin B resistance in clinical isolates of Leishmania donovani

Bidyut Purkait1, Ruby Singh1, Kirti Wasnik2, Sushmita Das3, Ashish Kumar1, Mark Paine4, Manas Dikhit1,Dharmendra Singh1, Abul H. Sardar1, Ayan K. Ghosh1 and Pradeep Das1*

1Department of Molecular Parasitology and Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Indian Council ofMedical Research, Agamkuan, Patna, 800007 Bihar, India; 2Department of Biotechnology, National Institute of Pharmaceutical Educationand Research, Hajipur, Bihar, India; 3Department of Microbiology, All India Institute of Medical Sciences, Patna, India; 4Department of

Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK

*Corresponding author. Tel: +91-0612-2631565; Fax: +91-0612-2634379; E-mail: [email protected]

Received 14 April 2013; returned 31 May 2014; revised 24 October 2014; accepted 30 November 2014

Objective: Silent information regulator 2 (Sir2) is involved in parasite survival and apoptosis. Here, we aimed toexplore the involvement of Sir2 in amphotericin B (AmB) resistance mechanism in Leishmania donovani.

Methods: The expression levels of Sir2, MDR1 and NAD+ biosynthetic pathway enzymes in AmB-resistantand -susceptible parasites were measured and total intracellular NAD+/NADH ratios were compared.Overexpression and knockout constructs of Sir2 were transfected in AmB-resistant and -susceptible parasites.Both resistant and susceptible parasites were inhibited with sirtinol for 4 h. The deacetylase activity of Sir2,the expression level of MDR1, the rate of AmB efflux, concentrations of reactive oxygen species (ROS) and levelsof apoptosis were examined in WT, inhibited and transfected parasites, and the AmB susceptibility of the respect-ive parasites was measured by determining the LD50 of AmB.

Results: Levels of mRNA, protein and NAD+-dependent deacetylase activity of Sir2 were elevated in resistant versussusceptible parasites. Inhibition and/or deletion of Sir2 allele showed a decreased mRNA level of MDR1, lower drugefflux, increased ROS concentration, apoptosis-like phenomenon and decreased LD50 of AmB in resistant parasites.In contrast, Sir2 overexpression in susceptible parasites reversed drug susceptibility producing a resistantphenotype. This was associated with increased LD50 of AmB along with increased expression levels of MDR1,drug efflux and reduced concentrations of ROS, corresponding to decreased apoptosis of resistant to WT sensitive.

Conclusions: Sir2 plays a critical role in AmB resistance by regulating MDR1, ROS concentration and apoptosis-likephenomena and may be a new resistance marker for visceral leishmaniasis.

Keywords: visceral leishmaniasis, AmB resistance, Sir2, MDR1, ROS, apoptosis

IntroductionVisceral leishmaniasis (VL) or kala azar is caused by the protozoanparasite, Leishmania donovani, and is a symptomatic infection ofliver, spleen and bone marrow. The global estimates for the inci-dence and prevalence of VL cases are 0.5 and 2.5 million per year,respectively.1 In India, VL has been reported in parts of West Bengal,Uttar Pradesh and Bihar. It poses a particularly worrying healthproblem in the state of Bihar, accounting for nearly 90% of thetotal cases in India.2 Chemotherapy has been proven to be theonly effective way of controlling infections and is highly dependentupon antimony-containing drugs such as sodium stibogluconate(Pentostam). Nearly 64% of cases in hyperendemic regions ofBihar are now resistant to antimonials,3 and amphotericin B

(AmB) is used as the drug of choice in these antimony non-responsive zones of Bihar. AmB is a polyene antifungal drug oftenused intravenously for systemic fungal infections. It was originallyextracted from Streptomyces nodosus, a filamentous bacterium.4,5

The mechanism of action of AmB is associated with the formationof specific pores in the membrane as well as the formation of freeradicals by the auto-oxidation of AmB.6,7

Although, AmB chemotherapy has been proven to be verysuccessful in the treatment of VL in India, its extensive use isexpected to drive drug resistance,8 and indeed we have recentlyreported AmB-unresponsive cases in Bihar, India.9 Furthermore,we have shown that AmB resistance is associated with alterationsin the thiol metabolic pathway [responsible for AmB-induced react-ive oxygen species (ROS) detoxification], ABC transporter-MDR1

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(responsible for AmB efflux) and altered sterol profile of the mem-brane (responsible for impaired AmB binding and uptake).9

The biggest threat to the successful treatment of VL is thedevelopment of drug resistance, thus understanding the mechan-ism of AmB resistance is essential to combat the disease. It isknown that, while AmB triggers apoptotic cell death via ROS inAmB-susceptible field isolates of Leishmania, it fails to triggerapoptosis in resistant parasites.10

The SIR2 protein of yeast is the founding member of a large con-served family of proteins that are present in many organisms rangingfrom bacteria to humans.11 It is an NAD+-dependent histone deace-tylase (HDAC).12 The first evidence that the SIR2 proteins could beinvolved in functions other than transcriptional silencing camefrom the presence of the LmSIR2 (Leishmania major SIR2)-relatedprotein in cytoplasmic granules.13 Seven SIR2 genes were identifiedin humans, classified as sirtuins (SIRT1–7).14,15 Based on thesequence homology, the silent information regulator 2 (Sir2) familyis categorized as a type III family of HDACs.16 Histone and non-histone substrates are deacetylated by sirtuins in the presence ofNAD+ and lead to the production of the deacetylated protein, nico-tinamide and OAADPr (O-acetyl-ADPribose).17,18 Trypanosoma bruceiSIR2RP1 and also Sir2 of Leishmania exhibit both NAD+-dependentdeacetylase activity and ADP-ribosyltransferase activity.19,20 Sir2of Trypanosoma is involved in DNA-damage repair.19 Also inLeishmania, Sir2 is involved in parasite survival by preventingprogrammed cell death.21 Another enzyme, poly(ADP-Ribose)polymerase-1 (PARP1), is the major consumer of NAD+ and accountsfor more than 90% of poly(ADP-ribose) production.22,23 Therefore,Sir2 and PARP1 activity are interlinked.24

Sir2 is also involved in detoxification of the ROS and overexpres-sion of the Sir2 homologue; SIRT1 increases MDR1-induced drugefflux in drug-resistant cells.25 – 27 We therefore predicted that,in addition to other mechanisms of resistance,9 Sir2 may play asimilar role in regulating the expression of MDR1 and therebyAmB efflux from the resistant parasite.

Here, our aim was to find out the role of Sir2 in AmB resistancein L. donovani. We demonstrate for the first known time that theincreased expression of Sir2 in resistant parasites results inincreased efflux of AmB from the parasites by the activation ofMDR1 expression. Sir2 also suppresses the AmB-induced ROS,thus preventing AmB-induced apoptosis-like phenomena.

Materials and methods

Clinical isolatesTwo clinical isolates (used in our previous study)9 of VL obtained from thesplenic aspirates of AmB responder and non-responder patients of theIndoor Ward facility of the Rajendra Memorial Research Institute ofMedical Sciences, Patna, India,9 were grown in RPMI-1640 (Gibco) medium(pH 7.4), supplemented with 10% FBS (Gibco) and 1% penicillin (50 U/mL)and streptomycin (50 mg/mL) solution (Sigma) at 258C and maintainedfurther under drug pressure. We also collected and used additional twoAmB-resistant (R2 and R3) and one susceptible (S2) parasite for this study.

Characterization and clonal selection of clinical isolatesThe isolated strains were confirmed as L. donovani spp. by amplification ofkinetoplast DNA (kDNA) using a kDNA-specific primer (F, 5′-TCTGTGGCCCATTTGTTGTA-3′, and R, 5′-CATTTTTGGGTTT T CGGAGA-3′)9 and were furtherclonally selected in NNN-blood agar medium,9 and passed through

mice. At least 10–15 passages of different isolates were carried out in dif-ferent media. Moreover, all of the parasites used were of the same passagenumber. The cultures were tested for resistance/susceptibility by bothin vitro and ex vivo drug susceptibility assay as described earlier,9 andtheir respective LD50 values for AmB were determined. LD50 values forR2, R3 and S2 were similarly determined.

Semi-quantitative RT–PCR and real-time PCRReverse transcription was performed as described previously.9 The synthe-sized cDNAs were amplified by PCR for specific genes, i.e. Sir2, MDR1, PARP1,nicotinamidase, Na phosphoribosyltransferase, NaMN adenyltransferaseand ammonia-dependent NAD+ synthase. The products were run on 2%agarose gel, stained with ethidium bromide and finally documented andquantified using the Bio-Rad Gel Documentation System and associatedQuantity One software. The RT–PCR product was normalized with respectto the alpha-tubulin (A-tub) RT–PCR product. Semi-quantitative data werevalidated by quantitative real-time PCR as described previously in theLightCyclerw 480 (Roche).9 Primers used for both semi-quantitative RT–PCRand quantitative real-time PCR are summarized in Table S1, available asSupplementary data at JAC Online.

Measurement of NAD1/NADH ratioTotal intracellular NAD+/NADH ratios in resistant and susceptible parasiteswere measured using an NAD+/NADH quantification kit (BioVision) accord-ing to the manufacturer’s protocol. Data were recorded at 450 nm using aspectrophotometer (Hitachi, Japan).

Heterologous expression and purification of LdSIR2RP(recombinant L. donovani SIR2 protein) and antibodygenerationThe LdSIR2RP-encoding sequence (GenBank accession number JN621326)from L. donovani genomic DNA was amplified by PCR using a forward pri-mer (5′-GGGGGATCCATGACAGCGTCTCCGAGAGC-3′) and a reverse primer(5′-GGGAAGCTTTCACCTCTCATTCGGCGCC-3′). The amplified product of1122 bp was cloned into expression vector pET28a (Novagen) and thefusion protein LdSIR2RP was produced in Escherichia coli BL21 (DE3) byinduction with 0.8 mM IPTG for 4 h at 308C. The induced bacterial pelletwas resuspended in a buffer containing 100 mM Tris/HCl (pH 7.6), 2% SDSand 1 mg/mL lysozyme, and disrupted by sonication. The clarified super-natant was applied to an Ni-NTA–agarose column (Qiagen) and the recom-binant LdSIR2RP protein was eluted with 200 mM imidazole. The elutedfractions were checked for the presence of recombinant LdSIR2RP usingSDS–PAGE and western blot using anti-his antibody. Anti-LdSIR2RP anti-body was generated in rabbit by injection of purified Sir2.

Western blot analysisThe expression level of SIR2 protein was estimated by western blot ana-lysis using anti-SIR2 antibody. Briefly, total protein from 2×106 cells ofboth AmB-resistant and -susceptible parasites was electrophoresed onSDS–PAGE and transferred to nitrocellulose membrane; immunoreactivepolypeptide was detected using polyclonal anti-SIR2 antibody. The inten-sity of bands was analysed using Quantity One software (Bio-Rad).

Preparation of overexpression construct of LdSir2 andtransfection in LeishmaniaThe LdSir2 ORF was PCR amplified from a pCRw 2.1-TOPO TA-LdSir2 construct(generated in our laboratory) using primers with restriction sites HindIII andBamHI (underlined) (F, 5′-GGGAAGCTTATGACAGCGTCTCCGAGAG C-3′; R,5′-GGGGGATCCTCACCTCTC ATTCGGCGCC-3′) and cloned into Leishmania

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expression vector pLp-NEO2 (a kind gift from Dr Greg Matlashewski,Department of Microbiology and Immunology, McGill University,Montreal, Canada) in between HindIII and BamHI sites. Clone pLp-NEO2-LdSir2 (ORF in the right orientation) was transfected intoLeishmania parasites by electroporation using a Gene Pulser (Bio-Rad)under conditions described by Ashutosh et al.,28 to generate Sir2-overexpressed (OE) lines. Parasites transfected with the empty vectorwere taken as controls. Transfectants were selected and maintained inthe presence of 40 mg/mL G418 and further selected in 80 mg/mL G418.

Molecular constructs for knocking out LdSir2 allelesA 0.85 kb flanking sequence upstream (5′F) and 1.0 kB flanking sequencedownstream (3′F) of LdSir2 were PCR-amplified from L. donovani genomicDNA using the following primers: primer 1 (containing HindIII site, under-lined; 5′-CGAAAGCTTAG CACGATGCGTGACTCC-3′); primer 2 (containing SalIsite, underlined; 5′-ACGCGTC GACCAGGAGCGCCTCATCCTC-3′); primer 3(containing SmaI site, underlined; 5′-TGAT CCCGGGATCGGGAAGGTCGTAGAC-3′); and primer 4 (containing BglII site, underlined; 5′-GAAGATCTCTGCAGTCGCAT AAGCAC-3′). Primers 1 and 2 were used to amplify 5′F and primers 3and 4 were used to amplify 3′F; 5′F and 3′F were then cloned in pX63NEO andpX63HYG vectors (a kind gift from Dr A. Jardim, Institute of Parasitology,McGill University, Montreal, Canada) in between HindIII and SalI as well asSmaI and BglII sites to generate 5′F-pX63HYG-3′F and 5′F-pX63NEO-3′F con-structs. Both of these circular constructs were then digested with HindIII andBglII to generate linear constructs for transfection in Leishmania.

Generation of LdSir22/1 parasitesTo generate LdSir2 double-knockout (Sir22/2) L. donovani, transformationsof all of the constructs were performed by electroporation with a Bio-RadGene Pulsar apparatus using 450 V and 550 mF capacitance.29 Briefly, latelog phase promastigotes (1.0×107), harvested and washed twice in elec-troporation buffer (21 mM HEPES, 137 mM NaCl, 0.7 mM NaH2PO4, 6 mMglucose, pH 7.4), were finally resuspended in 500 mL of electroporationbuffer and taken in a 0.4 mm ice-chilled electroporation cuvette.A 50 mg aliquot of linear gene replacement cassette LdSir2:5′F-pX63NEO-3′F, dissolved in 40 mL of electroporation buffer, was thenadded to the cuvette and incubated on ice for 10 min followed by electro-poration. Cells were incubated further for 10 min on ice and added to 20 mLof drug-free RPMI-1640 medium. After 24 h of revival, 25 mg/mL G418 wasadded to the cuvette, which was kept at 248C for another 10 days withmild shaking. Finally, all of the transfected cells (LdSir22/+ mutants) weremaintained at 50 mg/mL G418. The latter was selected for the secondround of targeting using the LdSir2: 5′F-pX63HYG-3′F construct.

Plasmid complementationLdSir22/+ parasite was transfected with the pXG-PHLEO-LdSir2 plasmid (pre-pared by cloning LdSir2 ORF in pXG-PHLEO in BamHI site; pXG-PHLEO vectorwas kind gift from Dr Stephen M. Beverley, Department of MolecularMicrobiology, Washington University in St Louis, USA) and selected with min-imal doses of phleomycin (25 mg/mL). Finally, it was grown in the presenceof 50 mg/mL of drug. Then the second round of targeting using the LdSir2:5′F-pX63HYG-3′F construct was performed and the complemented (CM)transfectants (LdSir22/2-pXG-PHLEO) were maintained in RPMI-1640 con-taining 50 mg/mL G418, 50 mg/mL hygromycin B and 50 mg/mL phleomy-cin. The presence of episome in CM clones was determined by selectivelyamplifying LdSIR2 gene using a specific pXG-PHLEO backbone primer(5′-CCTCCCCCTGTCCCCGGG-3′) and an LdSir2 reverse one.

Modelling and dockingThe full-length amino acid sequence of SIR2 was retrieved from NCBI(AEO62168.1) with a predicted molecular mass of 40.49 kDa. The

structural data of 1J8F, 1Q14, 1ICI and 1S5P (PDB ID) were used as tem-plates and the alignment was manually edited to predict the tertiary struc-ture of LdSIR2. Five models were generated by Modeller 9v10. Modellerimplements comparative protein structure modelling by satisfaction ofspatial restraints and performs optimization of various models of proteinstructure with respect to a flexibly defined objective function.30 The targetmodel with the lowest DOPE score was further refined by loop modellingand side chain refinement by Discovery Studio v2.5 (Accelrys Software Inc.,San Diego, CA, USA) to increase the Profile3D score. The modelled structurewas set for validation by SAVES (http://nihserver.mbi.ucla.edu/SAVES/) andProSA-web servers.31 The final modelled structure was simulated in expli-cit solvent using the GROMACS v4.0.3 (the Groningen Machine for ChemicalSimulations) package with Gromos43a1 force-field parameters.32 Themodel was subjected to position-restrained molecular dynamic (MD)with an NPT ensemble, keeping the number of particles (N), system pres-sure (P) and temperature (T) as constant parameters for a 100 ps timeperiod. The final MD was carried out for 15000 ps (15 ns) under particlemesh Ewald33 electrostatistics in NPT conditions. Molecular docking ofthe compound (sirtinol) in the active site of this LdSir2 homology modelwas carried out using FlexX and GOLD software.34,35

Inhibition assay with sirtinolSirtinol, an inhibitor of Sir2 was added at a concentration of 15 mM to WTand transfectants and incubated for 4 h at 238C in a Biological OxygenDemand (BOD) incubator. The parasites were subsequently washed withPBS (pH 7.2) and treated with AmB. The LD50 values of AmB were thendetermined.

Enzyme activity of Sir2 and PARP1Total intracellular NAD+-dependent deacetylase activity of Sir2 and PARP1activity was measured for WT, sirtinol-treated, LdSir22/+ and OE parasites.Sir2 activity was evaluated using a SIRT1/Sir2 deacetylase fluorometricassay kit (CycLex). Briefly, total cellular extracts were prepared and usedas a cofactor for purified LdSir2RP. In the control reaction, the NAD+ ofthe kit was used as a cofactor for the purified LdSir2RP. The results wererecorded using an LS55 spectrofluorometer (Perkin Elmer). The resultswere expressed as the rate of reaction for the first 20 min, when therewas a linear correlation between the fluorescence and the period oftime. Similarly, PARP1 activity was measured before and after AmB treat-ment using a PARP1 enzyme activity assay kit (Millipore).

Drug efflux measurementLeishmania promastigotes (WT, sirtinol-treated, LdSir22/+ and OE para-sites) treated with AmB were harvested and transferred to fresh, drug-freemedium after different timepoints and incubated for 2 h prior to extrac-tion. The AmB efflux was measured using HPLC (Hitachi) in a manner simi-lar to that described earlier.9

MTT assay and determination of LD50 valuesThe MTTassay is a quantitative colorimetric assay for the measurement ofmetabolically active cells. This assay was performed to determine the LD50

values of AmB for the WT, sirtinol-treated, LdSir22/+ and OE parasites afterAmB treatment as described previously.9

Detection of accumulation of ROSIntracellular oxidant levels were determined using 2′,7′-dichlorodihydrofluorescein di-acetate (H2DCFDA) (Sigma), which is oxidized insidethe cell to the fluorescent dichlorofluorescein. Intracellular concentrationsof ROS of WT, sirtinol-treated, LdSir22/+ and OE parasites after AmB

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treatment were measured according to our previous work using an LS55spectrofluorometer (Perkin Elmer) with excitation at 504 nm and emissionat 529 nm.9 The accumulation of ROS has also been measured in the pres-ence of the ROS scavenger 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Sigma).9

Detection of phosphatidyl serine exposureThe annexin V–fluorescein isothiocyanate (FITC) conjugate was usedin conjunction with the PI to discriminate among the necrotic (annexinV–FITC and PI positive), apoptotic (annexin V–FITC positive and PInegative) and live (annexin V–FITC and PI negative) cells. WT, sirtinol-pretreated resistant, LdSir22/+ resistant and OE susceptible parasites afterAmB treatment were assessed for binding with annexin V–FITC and PI usingthe Annexin-V–FLUOS staining kit (Roche) following the manufacturer’sprotocol. The percentage of apoptotic cells was determined by flowcytometry using FC500 software in a Cytomix FC 500 (Beckman Coulter).A total of 10000 events were acquired for analysis. FL-1 log (x axis,FITC-fluorescence) versus FL-2 log (y axis, PI) were recorded and displayed.

DNA fragmentation analysisDNA fragmentation analysis was performed taking WT, sirtinol-pretreatedresistant, LdSir22/+ resistant and OE susceptible parasites after AmBtreatment. A total of 5×106 cells were harvested by centrifugationat 2000 g, and the cell pellet was resuspended in 500 mL of digestion buf-fer [10 mM EDTA (Sigma), 50 mM Tris, at pH 8 (Sigma), 0.5% (w/v) sodiumL-sarcosinate (Sigma), 0.5 mg/mL proteinase K (Sigma)] and kept at 48C for2–3 h. RNaseA (Roche) was added and incubated at 558 C for 1 h. An equalvolume of 1:1 phenol/chloroform (Merck) was added to the samples andcentrifuged at 14000 g for 5 min and to the collected aqueous layer50 mL of 3 M sodium acetate (pH 4.5; Sigma) and 2.5 volumes of 100%ethanol (pre-chilled) were added. The samples were kept overnight at2208C, then centrifuged at 14000 g for 5 min at 48C. The air-dried pelletwas then resuspended in TE buffer, pH 8.0. The quantity of DNA in eachsample was determined spectrophotometrically (Hitachi, Japan) and ali-quots of the DNA were electrophoresed in 1.5% agarose (Sigma) gel tosee the fragmentation of DNA.

Detection of metacaspase-like protease activity

Metacaspase-like protease activity in WT, sirtinol-treated resistant,LdSir22/+ resistant and OE susceptible parasites after AmB treatmentwere measured using Boc-GRR-AMC substrate as described previously.36

Antipain was used as an inhibitor of metacaspase.

Membrane integrity assayThe membrane integrity assay of WT, sirtinol-pretreated resistant,LdSir22/+ resistant and OE susceptible parasites after AmB treatmentwas performed as described previously by our group.37 Briefly, treatedand untreated parasites (1×106 cells/mL) were incubated for 3–9 h andrelease of lactate dehydrogenase from the cells with damaged membranewas measured by CytoTox assay kit (Promega) following the manufac-turer’s instructions. 1% Triton-X 100-treated promastigotes for 15 minwas taken as positive control.

Macrophage infection assayMacrophage infection assay (ex vivo assay) was performed according toour previous work with some modifications.9 Briefly, 104 THP1 cells (trea-ted with PMA–phorbol myristate acetate) per well on glass coverslips wereseeded. Once macrophages had adhered, Leishmania promastigotes (WT,LdSir22/+ resistant, OE susceptible parasites) were added at a ratio of 1:10(macrophage:parasite) and maintained at 378C in 5% CO2 for 6 h.Non-internalized promastigotes were eliminated and different concentra-tions of AmB were added to the wells and incubated for 48 h. Cells werethen fixed and Giemsa stained, and LD50 values were then calculated asdescribed previously.9 For parasite load determination,37 infected macro-phages were treated with 1.80 mg/mL AmB (LD50 of resistant strain).

Statistical analysisThe LD50 values of drugs were calculated by regression obtained throughprobit analysis of log dose–response data on the drug.38 All experimentswere conducted at least in triplicate and the results are expressed asmean+SD of three experiments. The data were statistically analysedusing the single ANOVA test. A P value of ,0.05 was considered significant.

Results

Resistance/susceptibility of parasites

The parasites isolated from the splenic aspirates of VL patients wereconfirmed as Leishmania spp. by kDNA PCR (Figure S1).9 They wereaxenic, as they were clonally selected in NNN-blood agar medium.9

The LD50 values of resistant and suceptible parasites determined byin vitro and ex vivo drug susceptibility assay are summarized inTable 1. The LD50 value of AmB was �7.2-fold higher in resistantcompared with susceptible parasites (Table 1). AmB-resistant clin-ical isolates showed no significant cross-resistance towards other

Table 1. Reversion of resistant and susceptible phenotype by inhibition, overexpression and deletion of one Sir2 allele (LdSir22/+) in in vitro and ex vivo(macrophage infection) AmB susceptibility assay

Experiments

LD50 value (mg/L) Fold changea

in vitro ex vivo in vitro ex vivo

WT resistant+AmB 0.80+0.014 1.80+0.025 — —WT susceptible+AmB 0.11+0.011 0.25+0.050 — —Sirtinol+resistant+AmB 0.21*+0.042 — �3.8 (�) —Sir22/+ resistant+AmB 0.20*+0.038 0.48*+0.035 �4.0 (�) �3.8 (�)OE susceptible (80 mg/mL G418)+AmB 0.41*+0.021 0.90*+0.028 �3.7 (�) �3.6 (�)

LD50, effective dose 50 means the concentration of drug at which 50% cells will die or 50% cells will be viable. Asterisks denote the data that aresignificantly different from WT resistant and susceptible strain, P,0.05.aFold increase or decrease from the LD50 value of the corresponding WT susceptible and resistant strains.

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structurally unrelated drugs, such as hexadecylphosphocholine,sodium antimony gluconate, paromomycin and sodium stibogluco-nate (not shown). The LD50 values of R2, R3 and S2 lines are sum-marized in Table S2.

Higher expression level of Sir2 and NAD1 biosyntheticpathway enzymes in AmB-resistant parasites

The expression level of Sir2 was investigated in stationary-phasecultures of AmB-resistant and -susceptible parasites. mRNA and

protein concentrations of Sir2 were found to be �3.5-fold and�3.0-fold higher in the resistant strain compared with the suscep-tible strain, respectively (Figure 1a–c). The expression of Sir2 wasalso found to be higher in R2 and R3 isolates compared with the S2line as determined by western blot (Figure 1d).

The mRNA level of the enzymes involving in NAD+ biosynthesisand regulating the Sir2 activity was found to be up-regulated(expression levels of nicotinamidase, Na phosphoribosyltransfer-ase, NaMN adenyltransferase and ammonia-dependent NAD+

synthase were found to be �3.0-, �2.1-, �2.0- and �2.5-fold

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Figure 1. Expression level of Sir2 in both resistant and susceptible parasites. (a) The mRNA level of Sir2 in resistant and susceptible parasites usingsemi-quantitative RT–PCR. A-tub PCR product was used as loading control. Panel 1 shows the gel images; panel 2 is a graphical representation ofthe densitometry data. (b) Quantitative real-time PCR of expression level of Sir2 in resistant and susceptible parasites. (c) Western blot of Sir2expression in total protein extracts of resistant and susceptible parasites using rabbit polyclonal anti-rLdSir2 antibody. Panel 1, blot image; panel 2,graphical representation of densitometry data. (d) Sir2 expression level of R2, R3 and S2 strains through western blot (e, gel images; and f, graphicalrepresentation of the densitometric data). Determination of mRNA level of NAD+ biosynthetic pathway enzymes [nicotinamidase (PNC),Na phosphoribosyltransferase (NPT), NaMN adenyltransferase (Ade transferase), NAD synthase] in AmB-resistant and -susceptible strains usingsemi-quantitative RT–PCR. (g) Determination of total intracellular NAD+/NADH ratio of both the resistant and susceptible strains. (h and i) mRNAlevel and the enzyme activity of PARP1. mRNA level was determined by semi-quantitative RT–PCR (h); panel 1, gel image and panel 2, densitometricanalysis data. (i) Activity of PARP1 was determined for the indicated parasites. Asterisks denote that the data are significant (P,0.05).

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up-regulated, respectively) in the resistant strain compared withthe susceptible strain (Figure 1e and f). The NAD+/NADH ratiowas also found to be higher in resistant parasites comparedwith susceptible parasites (Figure 1g).

LdSIR2RP is an NAD1-dependent deacetylase

The LdSIR2 gene-encoding sequence was cloned into the bacter-ial expression vector pET28a(+). The fusion protein with anN-terminal tail of six histidine residues was purified under non-denaturing conditions, to examine its enzymatic activity. Afterpurification and dialysis, the protein showed .90% purity as eval-uated by SDS–PAGE and Coomassie Blue staining (Figure 2a). Thepurified LdSIR2RP showed NAD+-dependent deacetylase activity(Figure 2c), consistent with other members of the SIR2 family,14,15

and the deacetylase activity was inhibited by sirtinol (inhibitor ofSir2) (Figure 2d).

Sirtinol interacts with Sir2

In order to elucidate the structural and functional relevance interms of substrate binding and specificity, a structural model ofSir2 was generated (for modelling and molecular dynamics simu-lation, see Figure S2) and sirtinol docked onto the active site using

FlexX (Figure 3b). The predicted binding mode of sirtinol in thebinding site of the protein showed hydrogen bonding interactionswith residues Glu-16, Asp-324 and Val-325 (Figure 3b). The hydro-gen atoms of amine groups form hydrogen bonds with the car-bonyl oxygen of Val-325 as well as the oxygen atom (O1) ofAsp-324. The hydrogen atom of the amide group also formshydrogen bonds with an oxygen atom of Glu-16 and an oxygenatom (both O1 and O2 atoms) of Asp-324 (Figure 3b). Similardocking interactions with all residues apart from Val-325 werepredicted with an alternative Gold Docking program (Figure 3a).Overall, the in silico predictions suggested that sirtinol coulddock into the active site, and thus could be used as a potent in-hibitor against L. donovani Sir2.

Confirmation of the generation of Sir2-OE andSir2-defective parasites

To see whether overexpression of LdSir2 would modulate the AmBsusceptibility of the susceptible parasites, the gene was OE in sus-ceptible L. donovani isolates. The presence and overexpression ofthe LdSir2 gene in transfectants (OE parasite) were confirmed byRT–PCR and western blotting (Figure 4a–c). The band intensityof the LdSir2 amplicon was �2.9- and 3.8-fold higher (through

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RT–PCR) and �2.8- and 3.6-fold higher (through western blot) intransfectants maintained at 40 and 80 mg/mL G418, respectively,compared with the WT susceptible one (Figure 4a–c), therebysuggesting that the LdSir2 transcript was present in high copynumbers in the transfectants maintained at 80 mg/mL G418.

To assess the biological functions of the Sir2 protein in modu-lating AmB-resistant properties, we attempted to create an LdSir2null mutant cell line by gene replacement. Leishmania are asexualdiploids and thus require two rounds of gene replacement.39 Wegenerated LdSir2-knockout constructs by gene replacement witheither hygromycin- or neomycin-selectable markers (Figure 4d).The double-knockout null mutants (LdSir22/2) did not survive(not shown). However, complementation with an episomalexpression of LdSir2 (Figure 4g) rescued the phenotype, and theCM clones (LdSir22/2 carrying pXG-PHLEO-LdSir2 expression con-struct) were propagated in the medium (Figure 4i). Deletion andreplacement of both alleles of LdSir2 from CM clones by Neoand Hyg genes were confirmed by PCR analysis on genomic DNA(Figure 4e) as well as by Southern blot analysis on Pst I-digestedDNA40 and PCR products (Figure S3; for materials and methodsand detailed results of Southern blotting, see Supplementarydata). Thus, one copy of the Sir2 gene is needed for the survivalof the parasite, consistent with previous studies.40 As expected,we found that single LdSir2 gene disruption (LdSir22/+) did notaffect the growth of the parasite (Figure 4i). We therefore investi-gated whether partial SIR2 deficiency (single LiSIR2 knockoutclones) might affect the resistance properties of the parasites.The single LdSir2 gene deletion was confirmed by PCR analysisof genomic DNA (Figure 4e), and western blot analysiswith anti-LdSir2 antibody; �40% LdSir2 in the LdSir22/+ line(Figure 4f) indicated that one of the LdSir2 alleles had been

knocked out of the transfectant that was resistant to neomycinor hygromycin.

mRNA level and enzyme activity of PARP1

The mRNA levels (Figure 1h) and enzyme activity (Figure 1i) ofPARP1 were investigated in both resistant and susceptible para-sites. The mRNA level of PARP1 was found to be �4.5-fold lowerin resistant parasites compared with the susceptible one(Figure 1h) after AmB treatment. Similarly, PARP1 activity wasfound to be �3.8-fold lower in the resistant parasites comparedwith the susceptible parasites (Figure 1i). However, after inhibition,as well as after deletion of one Sir2 allele (LdSir22/+), the mRNAlevel and activity of PARP1 were increased in resistant parasitescompared with WT resistant ones (Figure 1h and i), whereas inOE susceptible parasites, the mRNA level and activity of PARP1were decreased compared with WT susceptible ones after AmBtreatment (Figure 1h and i). PARP1 activity was found to belower in untreated resistant parasites compared with susceptibleparasites (Figure 1i).

Deacetylase activity of Sir2

The deacetylase activity of LdSir2 was found to be �2.5-foldhigher in resistant parasites compared with susceptible ones(Figure 2e). Following the inhibition and deletion of one Sir2 allele(LdSir22/+), deacetylase activity was decreased (Figure 2e). Incontrast, deacetylase activity was increased in OE susceptiblecompared with WT susceptible parasites (Figure 2e). Deacetylaseactivity was also decreased in LdSir22/+ R2, R3 lines and increasedin OE S2 line (Figure S4).

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Figure 3. Interaction of LdSir2 with sirtinol. Panel A shows the model of LdSir2 showing interaction with sirtinol predicted by GOLD software (interactingresidues: Glu-16 and Asp-324). Panel B shows the same interaction predicted by FlexX software (interacting residues: Glu-16 and Asp-324 and Val-325).This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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Sir2 regulates the MDR1 expression and drugefflux machineryPreviously we have reported that efflux of AmB, mediated byMDR1, is one of the mechanisms of AmB resistance.9 The mRNA

level of MDR1 and the rate of AmB efflux were higher in resistantparasites compared with susceptible ones (Figure 5a and b), asreported.9 After Sir2 inhibition as well as after deletion of oneSir2 allele (LdSir22/+), the MDR1 mRNA level of resistant parasites

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Figure 4. (a–c) Characterization of Sir2 OE susceptible (S) strain. (a) Determination of mRNA level of LdSir2 by semi-quantitative RT–PCR in OE S (maintainedat 40 and 80 mg/mL G418) and WT S parasite. A-tub PCR was used as loading control. Panel 1 shows the gel images; panel 2 is a graphical representation ofthe densitometric data. (b) The presence of pLp-NEO2-LdSir2 construct in transfected susceptible parasites was detected by PCR. Lane 1 shows the bandobtained by PCR using pLp-NEO2 vector-specific forward primer (designed from just upstream of the start code of LdSir2) and LdSir2 specific reverse primer.Lane 2 is negative control to show the PCR specificity. Lane M is a 1 kb marker. The figure shows that the transfected parasites contain LdSir2 ligated in adirect orientation in pLp-NEO2 vector. (c) Western blot analysis of Sir2 expression in total protein extracts of OE S (maintained at 40 and 80 mg/mL G418) andWT S parasites using anti-rLdSir2 antibody. Western blot shows that LdSir2 is present in high copy numbers in OE S parasites maintained at 80 mg/mL G418.Panel 1 shows the blot image and panel 2 is graphical representation of densitometric data. (d–i) Targeted gene replacement of LdSir2 alleles. (d) Schematicrepresentation of the LdSir2 locus and the plasmid constructs used for gene replacement. (e) Agarose gel analysis of PCR amplified products of LdSir2gene. Lanes WT, LdSir22/+ and CM (LdSir22/2 parasites transfected with pXG-phleoLdSir2 plasmid) correspond to PCR with gDNA from WT, LdSir22/+

and CM parasites, respectively, with external primers. External forward and reverse primers were generated from 42 bp upstream of LdSir2 gene andposition 36 downstream of the stop codon of the gene, respectively. The expected sizes of the LdSir2, HYG and NEO PCR products are 1.2, 3.5 and3.7 kb. (f) Panel 1, western blot result using anti-LdSir2 and anti-A-Tub antibody. Panel 2 shows the bar diagram depicting the band intensities in thewestern blot. Band intensity was quantified by Quantity-One Software (Biorad). (g) Presence of LdSir2 in CM parasites. Lanes 3 and 4 are Sir2 bands,PCR amplified from CM parasites, Lanes 1 and 2 are negative controls. Lane M is the marker. (h and i) PCR amplification of Hyg and Neo gene from CMparasites, confirming the successful deletion of two alleles of Sir2 by the hygromycin and neomycin cassette. (j) Growth curve kinetics of WT, LdSir22/+

and CM parasites indicating that one copy of Sir2 gene is essential for the survival of the parasites. Asterisks denote that the data are significant (P,0.05).

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was decreased and consequently the rate of efflux of AmB wasdecreased (Figure 5a and b) and the intracellular content ofAmB was increased (Figure 5c). However, the MDR1 mRNA leveland consequently the rate of AmB efflux were increased inSir2-OE susceptible parasites (Figure 5a and b). Similar resultswere obtained for R2 and S2 lines (Figure S5A–C), where geneticmanipulation of Sir2 influences MDR1 expression and, simultan-eously, AmB efflux from the parasites. This clearly suggests thatSir2 directly or indirectly regulates the expression of MDR1 andmodulates the MDR1 mediating AmB efflux, thereby regulatingthe AmB resistance phenomena.

Accumulation of ROS

The intracellular ROS production after AmB treatment was mea-sured using H2DCFDA. The ROS concentration in the susceptiblestrain was found to be significantly higher than that in thedrug-treated resistant strain (Figure 6a), as reported previously.9

Treatment with sirtinol increased the concentration of ROS in theresistant strain significantly (�3.1-fold) after AmB administration.Similarly, the concentration of ROS was also increased significantly(�3.2-fold) in LdSir22/+ resistant parasites compared with WTresist-ant parasites after AmB treatment (Figure 6a). In Sir2-OE susceptibleparasites, the ROS concentration was decreased (�2.5-fold) afterAmB treatment (Figure 6a). Similar variation in ROS concentrationwas also observed in R2, R3 and S2 lines (Figure S6). Treatmentwith the ROS scavenger significantly decreased the concentrationof ROS and this, in turn, increased the rate of parasite survival (notshown). Sirtinol had no significant toxic effect on untreated resistantand susceptible parasites (Figure S7).

Analysis of AmB-induced apoptotic-like phenomena aftersirtinol treatment and/or deletion of single allele of Sir2

AmB induced apoptosis-like phenomena in susceptible parasitesbut failed to induce apoptosis in resistant parasites,10 as

demonstrated by DNA fragmentation and phosphatidyl serine(PS) externalization (Figure 6b–d). We investigated whether sirti-nol inhibition as well as the deletion of one Sir2 allele could induceany apoptosis-like phenomena in resistant parasites and whetherSir2 overexpression could reverse the apoptotic phenomena of thesusceptible parasites after AmB treatment. Sirtinol could induceDNA fragmentation in Lesihmania,41 a hallmark of metazoanapoptosis.42 Agarose gel (1.5%) analysis showed an oligosomalladder of DNA after inhibition with sirtinol in resistant parasites(Figure 6b). Similarly, AmB could also induce oligosomal DNA deg-radation in LdSir22/+ resistant parasites (Figure 6b). On the otherhand, in susceptible parasites AmB could induce oligosomal DNAdegradation, but this was significantly reduced in OE susceptibleparasites (Figure 6b). Overall, this indicates that a single knockoutand inhibition of Sir2 induced DNA degradation in resistant para-site, and Sir2 overexpression reduced the level of DNA degradationin susceptible parasites after AmB treatment. Thus, LdSir2 may beinvolved in controlling AmB-induced DNA degradation and there-fore contribute to AmB resistance.

PS is normally confined to the inner leaflet of the cell mem-brane and is externalized when the cell is committed to apop-tosis.43 PS externalization was detected by annexin V labelling(Figure 6c and d). Flow cytometric analysis revealed that, afterinhibition with sirtinol, annexin V-positive cells increased �8-foldin resistant parasites compared with WT ones. Similarly, inLdSir22/+ resistant parasites, annexin V-positive cells (apoptoticcells) were increased �9-fold compared with WT ones (Figure 6cand d). In the susceptible parasites, AmB alone could induce theexternalization of PS (10% of apoptotic cells), whereas in the OEsusceptible parasites the percentage of annexin V-positive (apop-totic) cells (Figure 6c) was decreased �2.0-fold compared withthe WT susceptible one after AmB treatment (Figure 6d). Thesedata clearly indicate that both inhibition and single knockout ofSir2 induce apoptosis-like phenomena in AmB resistance para-sites and after SIR2 overexpression susceptible parasites becomeresistant to AmB-mediated apoptosis.

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Figure 5. Sir2 controls (directly or indirectly) the expression of MDR1 and thereby controls the AmB efflux from the parasites. (a) Determination of themRNA level of MDR1 in WT resistant (R), LdSir22/+ R and OE S parasites by semi-quantitative RT–PCR. A-tub PCR was as loading control. Panel 1, agarosegel image. Panel 2, densitometry scanning results of bands. (b) Efflux of AmB from AmB-treated WT R and S strains as well as LdSir22/+ R and Sir2 OE Sparasites. Extracellular AmB content was measured by reverse-phase HPLC. (c) Intracellular AmB content (also measured by HPLC) of the indicatedparasites was measured after AmB treatment for defined timepoint. Genetic manipulation of Sir2 influences the level of intracellular AmB content.Asterisks denote that the data are significant (P,0.05).

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Metacaspase-like protease activity was found in apoptotic cells(sirtinol-treated resistant, LdSir22/+ resistant and AmB-treatedsusceptible; Figure 6e). Sensitive promastigotes treated with mil-tefosine were considered as positive control and very high activityof metacaspase was observed (Figure 6e). During this apoptoticevent the membrane of the parasite was intact as determinedby lactate dehydrogenase assay (Figure 6f).

Sirtinol treatment and/or deletion of a single allele of Sir2influences the parasite load in infected macrophagesafter AmB treatment

We investigated the effect of genetic manipulation of Sir2 onparasite killing in infected macrophage after AmB treatment.Single knockout of Sir2 (in resistant parasite) significantly

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Figure 6. Determination of intracellular ROS and ROS-mediated apoptosis-like phenomena. (a) Generation of intracellular ROS at 24 h after AmBtreatment was measured for WT R, WT S, sirtinol-pretreated resistant, LdSir22/+ R and Sir2 OE S parasites. Untreated parasite was taken as control.(b) Genomic DNA (gDNA) degradation was investigated by analysing the isolated gDNA on 1.5% agarose gels for untreated and treated R and Sparasite. Lane 1, marker; lane 2, R; lane 3, R + AmB; lane 4, sirtinol+R+AmB; lane 5, Sir22/+ R+AmB; lane 6, S; lane 7, OE S+AmB; and lane 8,S+AmB. Deletion of one Sir2 allele (LdSir22/+) induces DNA degradation in resistant parasites and Sir2 overexpression reduces the level of DNAdegradation in susceptible parasites. (c) Assay of cell death taking the same set of parasites used in DNA degradation assay by flow cytometry. Thefigure denotes differential changes in apoptotic population (annexin V-positive but PI-negative; expressed as the percentage of apoptotic cells) inresponse to sirtinol, deletion of one Sir2 allele as well as Sir2 overexpression. (d) Graphical representation of percentage of apoptotic cells.(e) Metacaspase-like protease activity was determined. An increased fluorescence intensity indicates increased metacaspase-like protease activity.(f) Measurement of released lactate dehydrogenase to determine the membrane integrity. Asterisk denotes that the data are significant (P,0.05).This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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increased the antileishmanial effect of AmB and Sir2 overexpres-sion (in susceptible parasites) significantly inhibited the antileish-manial effect of AmB on infected macrophage (Figure 7 andFigure S8). Therefore, in the amastigote stage, Sir2 also influencesthe AmB susceptibility of the resistant and susceptible parasites.

Reversion of AmB-resistant phenotype by inhibition aswell as by deletion of one Sir2 allele

Sir2 inhibition with sirtinol demonstrated the reversion of the resist-ant property, as the LD50 of AmB for the resistant strain decreased�3.8-fold (Table 1). However, the LD50 of the susceptible strain wasnot altered by preincubation with sirtinol (not shown).

Similarly, the LD50 value of AmB for LdSir22/+ resistant parasiteswas decreased �4.0-fold (Table 1) compared with WT resistantones, indicating a reversion of the resistant phenotype. In macro-phage infection assays, we observed a decrease (�3.8-fold) inthe LD50 value of AmB for the LdSir22/+ resistant parasites com-pared with WT ones (Table 1), which again indicates the partialreversion of the resistant phenotype.

For R2 and R3 lines, we found similar types of variations in LD50

values (3–4-fold difference) after inhibition and/or after singlegene deletion of Sir2 (Table S2).

Overexpression of LdSir2 reverses susceptibility

After overexpression of SIR2, the LD50 value of AmB was increased(�3.7-fold) in OE susceptible parasites maintained at 80 mg/mLG418 (Table 1), as demonstrated by in vitro drug susceptibilityassay, indicating the reversion of the susceptible phenotype.Similarly, in macrophage infection assay the LD50 of AmB wasincreased �3.6-fold in OE susceptible parasites maintained at80 mg/mL G418 (Table 1) from the WT susceptible parasites.There was no significant change in the LD50 value of resistantparasites after overexpression of Sir2 (not shown). In the S2 line,we found a similar type of variation in LD50 values after Sir2 over-expression (Table S2).

DiscussionThe present study aims to understand the involvement of Sir2in AmB resistance in L. donovani parasites. The resistant and

susceptible nature of all the parasites was confirmed by in vitroand ex vivo drug susceptibility assays (Table 1 and Table S2). Thehigher expression level (3–4-fold) of LdSir2 in all AmB-resistantparasites compared with the susceptible ones (Figure 1) was cor-related with a recent similar report on a miltefosine-resistantL. donovani strain,44 emphasizing an important role for Sir2 inAmB resistance.

In order to characterize the Sir2-dependent mechanism ofAmB resistance, we examined clinical isolates of L. donovani byinhibition of Sir2, as well as generation of LdSir22/+ resistant para-sites and Sir2 OE susceptible parasites. Sirtinol was used as aninhibitor of Sir2 to analyse the effect of Sir2 inhibition on AmBresistance. The in silico modelling and docking studies revealedthat sirtinol could specifically inhibit LdSir2. The FlexX Dock(221.81) and GOLD (46.18) fitness scores indicate a strong inter-action between the ligand (sirtinol) and target protein (LdSir2).Whilst one must treat such predictive modelling with caution,this nevertheless suggests that sirtinol could be used as a potentinhibitor against the Sir2 protein of L. donovani.

Sir2 activity is dependent on NAD+, as evident by the highactivity of Sir2 correlating with a high NAD+/NADH ratio(Figure 1).13,14,25 Furthermore, up-regulation of NAD+ biosyntheticpathway enzymes was evident in resistant parasites (Figure 1eand f), indicative of high NAD+ concentrations compared withsusceptible parasites. PARP1 is a major consumer of NAD+.22,23

Moreover, our inhibition, overexpression and partial deletion stud-ies showed that the LdSir2 activity was inversely proportional toPARP1 activity. Therefore, PARP1 and Sir2 activity may be inter-linked through NAD+ concentration in Leishmania. Although theNAD+ concentration in the resistant parasites was higher thanin susceptible ones, the mRNA levels and activity of PARP1remained lower. This suggests that the resistant parasite dam-pens PARP1 activity, thus increasing NAD+ availability for Sir2activity (Figure S9), and this phenomenon may also be due tothe ability of Sir2 to deacetylate the PARP1 and inactivate it.45

To gain insight into the Sir2-mediated AmB resistance, weinvestigated whether the molecule might directly or indirectlyregulate the expression of MDR1 and MDR1-mediated AmBefflux.9 Sir2 inhibition and/or partial deletion of Sir2 resulted indecreased mRNA level of MDR1 along with decreased drug efflux,whereas Sir2 overexpression increased the level of MDR1 along

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LdSi

r2–/

+ R +

Am

B R S

R + A

mB

S + A

mB

OE S

+ A

mB

LdSi

r2–/

+ R +

Am

B

300

Am

ast

igo

tes/

10

0M

F

200

100

0

Figure 7. Cell viability of amastigotes after genetic manipulation of Sir2 in presence of AmB (at LD50 of resistant strain). Macrophages were infected with:WT R; LdSir22/+ R; WT S; OE S and subsequently treated with 1.80 mg/mL AmB for 48 h. (a) Graph showing the number of amastigotes in infectedmacrophage for the indicated parasites. (b) Graph showing the number of infected macrophage for the indicated parasites. Asterisks denote thesignificant changes with P,0.05.

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Sirtinol

(Sir2 inhibitor)Sir2

inhibition

Histone

hyperacetylation

Induced gene

expression

APOPTOSIS

SensitiveResistant

Cell survival

longevity

Gene

silencing

Enhanced Sir2

activity

Increased

NAD+

NAD

synthase

NaAD

NAm

Nicotinamidase

PhosphoribisyltransferaseAdenyltransferase

No DNA damage DNA damage

ROS

Auto-oxidation

AmB

AmB

Sir2 (of resistant)

activating

MDR1

MDR1

efflux of AmB

No PARP1

activation

PARP1

activation

Increased nicotinamide

decreased NAD

NaMN

Na

Histone

deacetylation

Figure 8. Hypothetical model showing the intracellular events involving Sir2 in AmB-resistance phenomena. In the resistant parasites intracellular AmBeffluxes from the parasites by Sir2-activated (directly or indirectly) MDR1. Remaining intracellular AmB auto-oxidizes to produce ROS and this ROS isdetoxified by a Sir2-dependent unknown mechanism and thereby Sir2 prevents the ROS-mediated apoptosis. Moreover, in resistant parasites higherSir2 activity is regulated by lower PARP1 activity and higher NAD+ availability as the NAD+ biosynthetic pathway enzymes are up-regulated. On theother hand, in susceptible parasites, expression level of Sir2 is low and, therefore, the expression level of MDR1 is low and consequently little or noAmB can be effluxed from the susceptible parasites. Therefore, intracellular AmB content for susceptible parasites is higher and the ROS produced byauto-oxidation of AmB induces apoptosis-like phenomena in the susceptible parasite. After inhibition (by sirtinol) and/or after partial Sir2 gene deletionof resistant parasites, MDR1 expression level is decreased and consequently, little AmB can be effluxed from the parasites; as a result, intracellular AmBcontent are increased and thereby ROS produced by auto-oxidation of a greater amount of intracellular AmB induces the apoptosis-like phenomena andthe resistant parasites become partially susceptible.

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with increased drug efflux (Figure 5), consistent with previouswork,27 demonstrating the involvement of Sir2 in regulatingthe expression and activity of MDR1 (directly or indirectly). Thesefindings provide strong evidence that expression of MDR1 plusMDR1-mediated AmB efflux can be controlled by HDAC Sir2, high-lighting the importance of chromatin remodelling in the regulationof drug resistance development.

The AmB-induced ROS generation pattern (Figure 6a) for resist-ant and susceptible clinical isolates was examined and found tobe higher in susceptible parasites, as described previously.9

Lower levels of AmB-induced ROS in resistant parasites wereincreased in sirtinol-treated and/or in LdSir22/+ resistant parasitesand, conversely, higher levels of AmB-induced ROS in susceptibleparasites were decreased after Sir2 overexpression. This suggeststhat AmB-resistant Leishmania, like Saccharomyces cerevisiae,25

induce an Sir2-mediated ROS scavenging mechanism, therebylowering the intracellular concentration of ROS and ultimatelyprotecting the cells from ROS-induced damage as the defenceresponse to the drug.

Consistent with previous findings,10 the biochemical evidenceshowed that AmB resistance in L. donovani is associated withreduced propensity to undergo drug-induced ROS-mediatedapoptosis, as demonstrated by PS externalization and DNA frag-mentation assays (Figure 6b–d). Sir2 inhibition induced apoptosis-like phenomena,41 and Sir2 overexpression protected againstapoptotic cell death.20,21 Therefore, Sir2 of L. donovani mayhave a role in regulating the AmB-induced apoptotic phenomenonin resistant parasites. We observed AmB-induced apoptosis inboth Sir2 inhibited resistant and in LdSir22/+ resistant parasites(Figure 6), whereas Sir2 overexpression reduced the level of DNAdegradation and PS externalization in susceptible parasites(Figure 6). Metacaspase-like protease activity was found to beassociated with apoptosis. These findings provide the evidencethat AmB-induced apoptotic cell death can be protected againstby Sir2 in resistant parasites.

Resistant and susceptible phenotypes of the parasites werechecked after genetic manipulation of Sir2 and after inhibitionof Sir2. It was observed that overexpression of LdSir2 conferred3–4-fold decreased susceptibility to AmB compared with the sus-ceptible WT, whereas deletion of one Sir2 allele conferred 3–4-foldincreased susceptibility to AmB for the resistant parasites com-pared with the WT resistant ones (Table 1 and Table S2), consistentwith a recent report that LdMAPK1 overexpression conferred a2–3-fold difference in susceptibility of parasites to Sb(III) orSb(V).38 Thus, from the clinical point of view, a 3–4-fold differencein LD50 value appears significant and LdSir2 is an important deter-minant of the AmB susceptibility of the parasite, confirmed bydetermining the parasite load in infected macrophage after geneticmanipulation of Sir2 (Figure 7).

Taken together, we conclude that Sir2-regulated drug effluxmachinery (MDR1) (directly or indirectly), ROS detoxification andprevention of AmB-induced apoptosis have a cumulative effectin conferring resistance against AmB to the Leishmania parasites(Figure 8). Sir2 directly or indirectly increases the expression ofMDR1 and, thereby, efflux of AmB from the resistant parasite. Asa result, the intracellular AmB content of the resistant strain is lowand concomitantly fewer ROS are generated. In the resistantparasite, Sir2 plays a key role in detoxifying ROS as well as in pre-venting AmB-induced apoptosis. Thus, Sir2 inhibition as well aspartial deletion of Sir2 allele causes reversal of resistance, while

conversely Sir2 overexpression in susceptibility parasites producespartial resistance to AmB. Drug resistance is a multifactorial phe-nomenon, and Sir2 is one of the key factors in conferring AmBresistance. Sir2 is currently being used as a potent drug targetfor a variety of parasitic diseases.46 Our results suggest that itmay be used for the rational design of new drugs against VLand as a resistance marker.

AcknowledgementsWe thank Dr Armando Jardim for donating Leishmania-specific vectors,pX63HYG and pX63NEO, for the knockout study. We are thankful toDr Greg Matlawski for giving the Leishmania-specific vector pLpNEO2 foroverexpression studies and also to Dr Stephen M. Beverley for givingpXG-phleo vector for the complementation study. We are also thankfulto Mr Debraj Saha for technical assistance.

FundingThis work was supported by the Indian Council of Medical Research, Indiaand University Grants Commission, India.

Transparency declarationNone to declare.

Supplementary dataSupplementary methods and results, Figures S1–S9 and Tables S1 and S2are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

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