Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance Rati Tandon,a Sharat Chandra,b Rajendra Kumar Baharia,a Sanchita Das,a Pragya Misra,a Awanish Kumar,a Mohammad Imran Siddiqi,b Shyam Sundar,c Anuradha Dubea Division of Parasitology, Central Drug Research Institute, Lucknow, Indiaa; Division of Structural and Molecular Biology, Central Drug Research Institute, Lucknow, Indiab; Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Indiac eishmania, a protozoan parasite, causes leishmaniasis, a group of diseases with clinical manifestations that range from selfhealing cutaneous and mucocutaneous skin ulcers to a fatal visceral form (visceral leishmaniasis [VL]). This disease imposes a significant burden of mortality and morbidity, affecting 12 million people in more than 88 countries in the tropical and subtropical zones of the world (1). Since antileishmanial vaccines are still under development, control of the disease is dependent mostly on chemotherapy. Among the available antileishmanial drugs, pentavalent antimonials (SbV) have been the first-line drugs for all forms of leishmaniasis for almost 7 decades (2). During the last decade, several novel formulations of conventional antileishmanials, as well as new drugs, including the oral agent miltefosine, became available or were under investigation. However, their widespread use in poor countries is hindered by their high cost and also concerns about toxicity and emergence of resistance (3). Knowledge of antimonial resistance mechanisms in Leishmania spp. has primarily emerged from the study of laboratory-generated drug-resistant cell lines (4). Different mechanisms for drug resistance, such as gene amplification and reduced accumulation of the active drug due to either decreased influx or increased efflux and unique parasite thiol metabolism, have been suggested (5). Recently, information regarding drug resistance has been obtained in antimonial drug-resistant field isolates. It has been suggested that natural antimonial drug resistance is multifactorial and mechanistically distinct from laboratory resistance (6). Microarray and proteomic approaches have been employed to iden- L June 2014 Volume 58 Number 6 tify the proteins involved in drug resistance. One such study done by our group through proteomics revealed six proteins upregulated in the membrane-enriched fraction and 14 proteins in the cytosolic fraction that were overexpressed in a sodium antimony gluconate (SAG)-resistant strain (LdSR) compared with those in a SAG-sensitive strain (LdSS) of Leishmania donovani (7). The major proteins in the membrane-enriched fraction were ATP-binding cassette (ABC) transporter, heat shock protein 83 (HSP-83), glycosylphosphatidylinositol (GPI) protein transamidase, cysteine-leucine-rich protein, and 60S ribosomal protein L23a, whereas in the cytosolic fraction, proliferative cell nuclear antigen (PCNA), the proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, and tubulin-beta chain have been identified. PCNA, an important protein involved in DNA replication and its damage and repair, has not been hitherto reported for its involvement in drug resistance in any parasite. This study demonstrates for the first time that the overexpression of L. donovani PCNA (LdPCNA) is associated with Received 24 August 2013 Returned for modification 6 December 2013 Accepted 28 February 2014 Published ahead of print 10 March 2014 Address correspondence to Anuradha Dube, anuradha_dube@hotmail.com. This is CSIR-CDRI communication no. 8629. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01847-13 Antimicrobial Agents and Chemotherapy p. 2997–3007 aac.asm.org 2997 Downloaded from http://aac.asm.org/ on October 15, 2017 by guest Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by >3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ⬃5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV (from 41.2 ⴞ 0.6 ␮g/ml to 66.5 ⴞ 3.9 ␮g/ml) and SbIII (from 24.0 ⴞ 0.3 ␮g/ml to 43.4 ⴞ 1.8 ␮g/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIII treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism. Tandon et al. resistance to sodium antimony gluconate in L. donovani field isolates. MATERIALS AND METHODS 2998 aac.asm.org Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on October 15, 2017 by guest Animals. Laboratory-bred male golden hamsters (Mesocricetus auratus) (45 to 50 g) from the Central Drug Research Institute’s (CDRI’s) animal house facility were used as experimental hosts. They were housed in a climate-controlled room and fed with standard rodent food pellets (Lipton India, Mumbai, India) and water ad libitum. The use of hamsters was approved by the Institute’s animal ethics committee. Parasites. L. donovani strain Dd8 (MHOM/IN/80/Dd8) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 U/ml penicillin (Sigma), and 100 mg/ml streptomycin (Sigma) at 26°C. L. donovani SAG-resistant LdSR (identification no. 2039), LdSR1 (1216), and LdSR2 (761) and SAG-sensitive LdSS (2001) strains drawn from splenic aspirates of VL patients from regions where leishmaniasis is endemic (Muzaffarpur, Bihar, and Banaras Hindu University [BHU], Varanasi, Uttar Pradesh, India) were grown in vitro in a biphasic medium and cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 0.2% NaHCO3, 2.05 mM L-glutamine, 12 mM HEPES buffer (HiMedia, India), 10% (vol/vol) heat-inactivated fetal bovine serum (HIFBS) (Gibco, Germany), and 50 mg/liter gentamicin at 25°C. These strains have also been maintained in hamsters through serial passage (i.e., from amastigote to amastigote) (8). In vitro sensitivity of clinical isolates to SbV and SbIII. The sensitivity of strain Dd8 and clinical isolates to SbV (SAG; Albert David) was investigated against intramacrophage amastigotes as described earlier (9). Briefly, the mouse macrophage adherent cell line J774A.1 (1 ⫻ 105 cells/ well) (Nunc) was cultured in 8-well chamber slides and infected with stationary-stage promastigotes at a ratio of 10:1 (L. donovani/macrophage) and was incubated in 5% CO2 for 12 h at 37°C. After being washed with serum-free medium, the cells were reincubated for 48 h with SAG (0, 5, 10, 20, 40, 80, 160, 320, and 640 ␮g/ml). Chamber slides were fixed in absolute methanol and stained with Giemsa stain. The numbers of amastigotes per cell were counted in 100 macrophages, and percent killing was calculated. The 50% inhibitory concentration (IC50) was obtained by plotting a graph of the percentage of inhibition at different concentrations of the formulations using Origin 6.1 version software. In addition, susceptibility of promastigotes against a trivalent antimonial (SbIII) (potassium antimony tartrate; Sigma) was assessed by the 2,3bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay (cell proliferation kit; Life Technologies). Briefly, 1 ⫻ 105 promastigotes in 200 ␮l/well were seeded in a 96-well tissue culture plate (Nunc) and incubated with 0, 5, 10, 20, 40, 80, 160, 320, and 640 ␮g/ml of drug for 48 h. After that, an XTT assay was performed as per the manufacturer’s protocol, and optical densities (OD) were obtained at 480 nm with the reference OD at 650 nm (OD650). Isolation of procyclic and metacyclic stages of the promastigotes. Procyclic and metacyclic stages of the promastigotes of stationary cultures were isolated by lectin-mediated agglutination using peanut agglutinin (PNA) (Sigma) as described earlier (10). Purification of splenic amastigotes from the infected hamsters. Splenic amastigotes were isolated using the Percoll (Sigma) density gradient centrifugation method, as described by Chang (11), from the aseptically isolated spleen of hamsters with well-established infections (45 to 60 days old). Cell line. The mouse macrophage cell line J774A.1 was maintained in RPMI 1640 through serial passage in 75-cm2 culture flasks (Nunc) at 37°C and 5% CO2. The confluent cells were harvested using a cell scraper. Preparation of soluble L. donovani promastigote and amastigote antigens. Promastigote and amastigote soluble antigens (soluble Leishmania donovani lysate [SLD]) were prepared as per the method described previously (12). The protein content of the supernatants was estimated by the Bradford method, and supernatants were stored at ⫺70°C. Cloning, expression, and purification of rLdPCNA. L. donovani genomic DNA was isolated from 108 promastigotes as described earlier (13). The PCNA gene was amplified using Taq polymerase (TaKaRa) lacking 3= to 5= exonuclease activity through PCR using PCNA-specific primers designed on the basis of the Leishmania major PCNA gene sequence (forward, 5=-CATATGATGCTCGAGGCTCAGGTCCAG-3=, and reverse, 5=-GGATCCCTCCGCATCGTCCACCTT-3=, with NdeI and BamHI restriction sites shown as underlined) in a thermocycler (BioRad) under conditions of 1 cycle of 95°C for 4 min, 30 cycles of 95°C for 1 min, 56°C for 30 s, and 72°C for 1 min, and finally 1 cycle of 72°C for 10 min. The amplified PCR product was electrophoresed in 1% agarose gel and eluted by GenElute columns (Qiagen). The eluted product was ligated in the pTZ57R/T (T/A) cloning vector (Fermentas) and transformed into competent Escherichia coli DH5␣ cells. The transformants were screened for the presence of recombinant plasmids with the recombinant LdPCNA (rLdPCNA) insert by gene-specific PCR under conditions similar to those previously mentioned. Isolated positive clones were sequenced (Chromous Biotech Pvt. Ltd., India) and submitted to the National Center for Biotechnology Information (NCBI). PCNA was further subcloned at the NdeI and BamHI sites in the bacterial expression vector pET28a (Novagen). The expression of rLdPCNA was checked in bacterial cells by transforming the PCNA-pET28a construct in the E. coli Rosetta strain. The transformed cells were inoculated into 5 ml Luria-Bertani medium and allowed to grow at 37°C in a shaker at 200 rpm. Cultures in the logarithmic phase (at an OD600 of ⬃0.5 to 0.6) were induced for 3 h with 1.0 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at 37°C. After induction, 1 ml cells was lysed in 100 ␮l sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 10% SDS, and 0.05% bromophenol blue, with 10% ␤-mercaptoethanol), and whole-cell lysates (WCL) were analyzed by 12% SDS-PAGE (13). The uninduced control cultures were analyzed in parallel. The overexpression of recombinant PCNA (rPCNA) was visualized by staining the gel with Coomassie brilliant blue R-250 (Sigma-Aldrich). For purification, 200 ml Luria-Bertani medium containing 50 ␮g/ml kanamycin was inoculated with the E. coli Rosetta strain transformed with pET28a-PCNA and grown at 37°C to an OD600 of ⬃0.6. Recombinant protein expression was induced by the addition of 1 mM IPTG, and the culture was incubated for an additional 3 to 4 h. The rLdPCNA was purified by affinity chromatography using Ni2⫹ chelating resin to bind the His6 tag fusion peptide derived from the pET28a vector. The cell pellet was resuspended in 4 ml lysis buffer (50 mM Tris-HCl [pH 8], 300 mM NaCl, and 20 mM imidazole) containing a 1:200 dilution of the protease mixture inhibitor (Sigma-Aldrich) and 1% Triton X-100, incubated for 30 min on ice with 1 mg/ml lysozyme (Sigma-Aldrich), and the suspension was sonicated for 10 20-s pulses (with 30-s intervals between each pulse) on ice. The sonicated cells were centrifuged at 15,000 ⫻ g for 30 min, and the supernatant was incubated at 4°C for 1 h with 1 ml nickel-nitrilotriacetic acid (Ni-NTA) Superflow resin (Qiagen, Hilden, Germany) previously equilibrated with lysis buffer. After being washed with buffer (50 mM Tris-HCl [pH 8] and 300 mM NaCl) containing different concentrations of imidazole (i.e., 20 and 50 mM), the purified rPCNA was eluted with elution buffer (50 mM Tris-HCl, 200 mM NaCl, and 250 mM imidazole [pH 7.5]). The eluted fractions were analyzed by 12% SDS-PAGE for purity. The protein content of the fractions was estimated by the Bradford method using bovine serum albumin (BSA) as the standard. Production of polyclonal antibodies against rLdPCNA and Western blot analysis. Purified rLdPCNA protein was used for raising antibodies in Swiss mice. Mice were first immunized using 25 ␮g of recombinant protein in Freund’s complete adjuvant. After 15 days, the mice were boosted three times with 10 ␮g of the recombinant protein each in incomplete Freund’s adjuvant at 2-week intervals, and serum was obtained by sacrificing the mice after the last booster. The antibody titer was determined by an enzyme-linked immunosorbent assay (ELISA). For immunoblots, purified recombinant protein, Leishmania promastigote wholecell antigens, and SLD were separated on 12% SDS-PAGE and Role of PCNA in SAG Resistance June 2014 Volume 58 Number 6 using GeneQuant (Bio-Rad). One microgram of the total RNA of each strain was used for the synthesis of cDNA with a first-strand cDNA synthesis kit (Fermentas). For real-time PCR, the following primers were designed using Beacon Designer software (Bio-Rad) on the basis of gene sequences available at NCBI: sense, 5=-GAGGTGACGATGGAGGAG-3=, and antisense, 5=-AGGTAGTAGCGAAGGTAGC-3=. Real-time quantitative PCR was carried out with 10 ␮l of SYBR green PCR master mix (Bio-Rad), 1 ␮g of cDNA, and primers at final concentrations of 300 nM in a final volume of 20 ␮l. PCR was conducted under the following conditions: initial denaturation at 95°C for 2 min followed by 40 cycles, each consisting of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s per cycle using the iQ5 multicolor real-time PCR system (Bio-Rad). All quantifications were normalized to the housekeeping gene ␣-tubulin. A no-template control and a no-reverse transcriptase control were included to eliminate contaminations or nonspecific reactions. The cycle threshold (CT) value was defined as the number of PCR cycles required for the fluorescence signal to exceed the detection threshold value (background noise). For calculation of differences in gene expression, ⌬CT values of PCNA were determined in the LdSR and LdSS strains, which indicates the differences between threshold cycles of PCNA and the housekeeping gene ␣-tubulin. After that, the difference between the ⌬CT value of PCNA of the SAG-resistant and -sensitive strains (⌬⌬CT) was calculated. The fold change in the expression of PCNA mRNA in the LdSR strain in comparison to that in the LdSS strain was determined by the formula fold change in expression ⫽ 2⫺⌬⌬CT. To confirm the role of PCNA in SAG resistance, the expression levels of LdPCNA were determined in another SAG-sensitive strain, Dd8 (also a reference strain), and in the SAG-resistant strains LdSR1 and LdSR2 by real-time PCR. Cloning of LdPCNA in pXG-=GFPⴙ and its overexpression in LdSS strain of L. donovani. The LdPCNA gene was amplified using the forward primer 5=-GGATCCATGATGCTCGAGGCTCAGGTCC-3= and the reverse primer 5=-GATATCCTCCGCATCGTCCACCTTCGC-3= (with BamHI and EcoRV restriction sites underlined) in a Thermocycler (BioRad) under conditions of 1 cycle of 95°C for 4 min, 30 cycles of 95°C for 1 min, 54°C for 30 s, and 72°C for 1 min, and finally 1 cycle of 72°C for 10 min. The amplified PCR product was ligated in the pTZ57R/T (T/A) cloning vector (Fermentas) and transformed into competent Escherichia coli DH5␣ cells. PCNA was further subcloned in vector pXG-=GFP⫹ (kindly donated by S. M. Beverley), creating plasmid pXG-=GFP⫹/PCNA. For overexpression of LdPCNA in the LdSS strain, promastigotes were transfected with pXG-=GFP⫹/PCNA using the low-voltage electroporation protocol as described elsewhere (20). Following electroporation, cells were transferred to 5 ml of complete RPMI 1640 medium and incubated at 26°C. G418 was added at different concentrations (0, 25, and 50 ␮g/ml) to the culture after 24 h. Overexpression of LdPCNA-GFP was confirmed by Western blot analysis of the cell lysate of the transfectants (LdSSPCNA-GFP) using anti-PCNA and anti-GFP antibodies (Roche). In addition to the pXG-=GFP⫹/PCNA plasmid, the pXG-=GFP⫹ vector was also transfected similarly, which served as a vector control (LdSS-GFP). Similarly, LdPCNA was also overexpressed in the SAG-resistant LdSR strain, and transfectants were maintained at different G418 concentrations (0, 25, and 50 ␮g/ml). In vitro assay for drug sensitivity. The wild type and transfectants growing at different G418 concentrations (0, 25, and 50 ␮g/ml) were analyzed for in vitro SbIII and SbV drug susceptibility similarly to the description above. Qualitative analysis of DNA fragmentation by SbIII. Wild-type LdSS and transfectant LdSS-GFP and LdSS-PCNA-GFP (at 50 ␮g/ml G418) promastigotes were treated with 30 ␮g/ml SbIII promastigotes for 24 h. Qualitative analysis of DNA fragmentation was performed by agarose gel electrophoresis of DNA extracted from 1 ⫻ 108 parasites as previously described (21). aac.asm.org 2999 Downloaded from http://aac.asm.org/ on October 15, 2017 by guest electrophoretically transferred onto a nitrocellulose membrane using a semidry blot apparatus (Hoefer SemiPhor; Pharmacia Biotech). After overnight blocking in 5% skimmed milk, the membrane was incubated with antiserum to the recombinant protein at a dilution of 1:1,000 for 120 min at room temperature (RT). The membrane was washed three times with phosphate-buffered saline (PBS) containing 0.5% Tween 20 (PBS-T) and then incubated in goat anti-mouse IgG horseradish peroxidase (HRP) conjugate solution at a dilution of 1:10,000 for 1 h at RT. The blot was developed by using diaminobenzidine-imidazole-H2O2. Glutaraldehyde cross-linking and size exclusion chromatography. For glutaraldehyde treatment, reaction mixtures with 50 to 100 ␮g of rLdPCNA in 20 mM phosphate buffer (pH 7.5) in a total volume of 100 ␮l were treated with 2 ␮l of 8% freshly prepared solution of glutaraldehyde for 2 to 5 min at 37°C. The reaction was terminated by the addition of 10 ␮l of 1 M Tris-HCl (pH 8.0). Cross-linked protein was solubilized by the addition of sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 10% SDS, and 0.05% bromophenol blue with 10% ␤-mercaptoethanol), and electrophoresis was conducted on an 8% SDS-PAGE gel. For size exclusion chromatography, approximately 500 ␮l (⬃500 ␮g) of purified recombinant protein was loaded onto a Superdex 200 10/300 GL column preequilibrated with buffer (50 mM Tris-HCl [pH 7.2], 200 mM NaCl) using a manual injector. Chromatography was performed on an AKTApurifier system (GE Healthcare) at a flow rate of 0.5 ml/min at 25°C, and the absorbance was monitored at 280 nm. The column was calibrated with standard molecular weight markers. In silico structure analysis of LdPCNA. In the absence of a crystal structure, the three-dimensional (3D) structure of LdPCNA was predicted by homology modeling using structural knowledge. The sequence of LdPCNA, consisting of 292 residues, was retrieved from the UniProt Knowledge Base (http://www.uniprot.org; UniProt accession no. B5TV91) and used as a query for NCBI BLASTP to search for suitable templates. Sequence alignment was performed between LdPCNA and template sequences using ClustalW (14) to identify conserved regions. The crystal structure of human PCNA (HuPCNA) (Protein Data Bank [PDB] accession no. 1VYM) (15) was used as a template for the construction of the LdPCNA model because it was found to be the most suitable template in terms of sequence identity and crystallographic resolution. The 3D model was generated using the software MODELLER (version 9.11) (16) with 1VYM as a template; the 2.3-Å resolved crystal structure of HuPCNA was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) database. A total of 20 models were generated for LdPCNA and rated according to the GA341 and DOPE scoring functions available with MODELLER. The refinement of the loop regions was done by using the “loopmodel” class available with MODELLER. The predicted 3D structures were evaluated using PROCHECK (17). Secondary structural analyses were performed with PDBsum (18) after modeling of the structure of the protein. All of the analysis and visualization of the structure files were done using Chimera (19). Assessment of differential expression of PCNA in different developmental stages of LdSS and LdSR strains of L. donovani. (i) By Western blotting. SLD (30 ␮g) from the log phase, stationary phase, and PNAisolated procyclic and metacyclic (10) stages of the promastigote and amastigote stages of LdSR and LdSS strains were subjected to SDS-PAGE separately on a 12% polyacrylamide gel and transferred to polyvinyl difluoride (PVDF) membranes. The membrane strips were blocked and incubated with the anti-PCNA primary antibodies and subsequently with anti-mouse IgG conjugated with HRP, and then the blots were developed using an ECL kit (Amersham). The images were scanned, and quantitative assessment was carried out by ImageJ software (NIH Image). The expression level was normalized to Grp78 prior to the calculation of the change in expression between the two strains. (ii) By real-time PCR. Real-time PCR was performed to assess the differential expression of mRNAs of LdPCNA in SAG-sensitive LdSS and SAG-resistant LdSR strains. Briefly, total RNA was isolated from different stages of the two strains using TRI reagent (Sigma-Aldrich) and quantified Tandon et al. RESULTS In vitro sensitivity of clinical isolates against SbV and SbIII. The in vitro SbV sensitivity was assessed in intramacrophage amastigotes in different clinical isolates, and the IC50 was calculated, which demonstrated that LdSS and Dd8 strains were sensitive while LdSR, LdSR1, and LdSR2 strains were resistant to SbV treatment. The sensitivity profile of the isolates determined by the SbIII promastigote assay correlated well with the SbV intracellular amastigote assay (Table 1). Cloning, expression, and purification of rLdPCNA. The LdPCNA gene was successfully amplified and cloned in the pTZ57R/T (T/A) cloning vector and transformed into competent DH5␣ cells (Fig. 1A and B). The positive transformants were sequenced and submitted to the National Center for Biotechnol- 3000 aac.asm.org TABLE 1 Clinical resistance profile and in vitro susceptibilities of field isolates of L. donovani to SbIII and SbV IC50 (mean ⫾ SD) (␮g/ml) for: Strain Response to SAG therapy SbV against intramacrophage amastigotes SbIII against promastigotes Dd8 LdSS LdSR LdSR1 LdSR2 Sensitive Sensitive Resistant Resistant Resistant 45.8 ⫾ 1.6 41.2 ⫾ 0.6 457.8 ⫾ 12.3 436.3 ⫾ 10.7 409.7 ⫾ 10.2 30.6 ⫾ 1.6 23.6 ⫾ 0.8 237.7 ⫾ 9.4 221.4 ⫾ 9.1 214.3 ⫾ 8.2 ogy Information (see above). The gene was further subcloned into the pET28a vector, and the recombinant plasmid was characterized with double enzymatic digestion (Fig. 1C) and checked for overexpression. An rLdPCNA overexpression band appeared with a predicted molecular mass of ⬃35 kDa. Comparisons of both noninduced and induced cultures in SDS-PAGE revealed that the protein was expressed successfully, and the induced protein band corresponded to its predicted size. The protein was purified to homogeneity by Ni-NTA affinity chromatography (Fig. 1D). rLdPCNA was used to raise a polyclonal antibody in Swiss mice, and the titer of the antibody produced was in the ratio of 1:25,600 as determined by an ELISA. The Western blot analysis with polyclonal antiserum to rLdPCNA recognized a single specific band of 32 kDa in both the whole-cell lysate and the soluble L. donovani lysate (SLD) (Fig. 1E). Glutaraldehyde cross-linking and size exclusion chromatography. Glutaraldehyde cross-linking and size exclusion chromatography confirmed the trimeric nature of the protein as a band of approximately 105 kDa appeared on SDS-PAGE of the crosslinked protein (Fig. 2A). A single elution peak at ⬃105 kDa represented the purified rLdPCNA on Superdex 200 column chromatography (Fig. 2B). In silico structure analysis of LdPCNA. HuPCNA is a toroidshaped homotrimeric protein, so to make a model of multichains together, the model-multichain-sym.py program of MODELLER is used. The LdPCNA has 39% sequence similarity (Fig. 3A) with the template, and the superimposition of the modeled complex with the template shows a root mean square deviation (RMSD) of 0.540 Å (Fig. 3B and C). The models generated in MODELLER were analyzed online by submission to the SAVES server (http: //nihserver.mbi.ucla.edu/SAVES_3/). For the final selected model, a Ramachandran plot generated by PROCHECK showed that 92.7% of the residues lie in the most favored regions (Fig. 3D), and 5.5%, 1.8%, and 0.0% of the residues lie in an additional allowed, a generously allowed, and a disallowed region, respectively. Secondary structure prediction was also performed with PDBsum to identify the location of the secondary structure (Fig. 3E). In the monomeric structure of LdPCNA, there are two structural domains, one with ␤-␣-␤-␤-␤-␣-␤-␤-␣-␤-␤-␤ and the other with ␤-␣-␤-␤-␤-␤-␤-␣-␤-␤-␤, joined by an interdomain connecting loop (IDCL). LdPCNA interacts with several proteins with the specific structural elements. Although most of the interactions of LdPCNA are mediated by the IDCL, the center loop is important for binding to cyclin D and the C terminus is important Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on October 15, 2017 by guest Determination of DNA synthesis in wild-type and transfectant promastigotes after treatment with SbIII. A 100-␮l portion of wild-type LdSS and transfectants (LdSS-PCNA-GFP and LdSS-GFP growing at a 50 ␮g/ml G418 concentration) in complete RPMI 1640 was plated in a 96well plate at 2 ⫻ 106 cells/ml and incubated with 100 ␮l of 30 ␮g/ml drug for 24 h, followed by bromodeoxyuridine (BrdU) labeling for 8 h. For a negative control, cells without added BrdU were also included. Newly synthesized DNA was detected using the BrdU cell proliferation assay (Calbiochem), following the manufacturer’s protocol for cell denaturation, mouse anti-BrdU antibody and HRP-tagged goat anti-mouse IgG applications. The chromogenic tetramethylbenzidine substrate was applied for 15 min, and the reaction was stopped with 2.5 N sulfuric acid. Absorbance was measured at 450 to 595 nm using a SpectraMax plate reader (Molecular Devices, Downingtown, PA). All samples were analyzed in triplicate in three independent experiments. Estimation of NO produced by macrophages, infected with wildtype and transfectant parasites, after treatment with SAG. The presence of nitrite (NO2⫺) content was measured using Griess reagent in the culture supernatants of J774A.1 macrophages infected with wild-type (LdSS and LdSR) strains and transfectants (LdSS-PCNA-GFP and LdSS-GFP) after treatment with SAG (30 ␮g/ml). Briefly, the macrophages were resuspended at 105 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum and plated in a 24-well plate (Nunc) and were allowed to adhere for 2 h in a CO2 incubator with a supply of 5% CO2 at 37°C. The wells were washed twice with serum-free medium, and the adherent macrophages were infected with a stationary phase of parasites at a ratio of 10:1 (Leishmania/macrophage) in 1 ml final solution of a complete medium and incubated overnight. After 16 h, free promastigotes were washed with serum-free medium, and infected macrophages were incubated with medium containing SAG at 37°C. Untreated infected macrophages and uninfected macrophages served as controls. Supernatant (100 ␮l) was collected from each well 12 h, 24 h, and 48 h after treatment with SAG, mixed with 100 ␮l of Griess reagent, and incubated for 10 min. The OD of the reaction was taken at 540 nm (22). Statistical analysis. The results (pooled data from three independent experiments) were analyzed by a one-way analysis of variance (ANOVA) test, and comparisons with control data were made with a Tukey posttest using the GraphPad Prism software program. A one-way ANOVA statistical test was used to assess the significance of the differences among various groups, and a P value of ⬍0.05 was considered significant. Ethics statement. Experiments on animals were performed after the approval of the protocol and following the guidelines of Institutional Animal Ethics Committee (IAEC) of the CDRI, which follows the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) under the Ministry of Forest and Environment, Government of India. Nucleotide sequence accession numbers. The LdPCNA gene sequence determined in this work has been deposited in GenBank under accession numbers FJ014501 and FJ014501.1. Role of PCNA in SAG Resistance confirmation in pTZ57R/T. Lane 1, 1-kb ladder; lane 2; NdeI- and BamHI-digested pTZ57R/T-PCNA. (C) Clone confirmation in pET28a. Lane 1, 100-bp ladder; lane 2, NdeI- and BamHI-digested pET28a-PCNA; lane 3, undigested plasmid. (D) Expression and purification of rPCNA in E. coli cells. WCL of transformed E. coli were separated on a 12% acrylamide gel and stained with Coomassie blue. Lane 1, molecular mass markers; lane 2, WCL before IPTG induction; lane 3, WCL after IPTG (1.0 mM) induction at 37°C; lanes 4, 5, and 6, wash fractions; lanes 7, 8, 9, and 10, eluted protein. (E) Western blot analysis using anti-rPCNA antibody in uninduced WCL, induced WCL, and Leishmania WCL and SLD. Lane 1, molecular mass markers; lane 2, uninduced E. coli WCL; lane 3, induced E. coli WCL; lane 4, purified rPCNA; lane 5, Leishmania WCL; lane 6, SLD. for binding to DNA polymerase ε and replication factor C (23). Monomeric LdPCNA is superimposed with HuPCNA and the Entamoeba histolytica PCNA monomer with RMSD of 0.490 Å and 0.986 Å (Fig. 3F). The structural architecture of the IDCL is similar to that observed in HuPCNA; it clearly shows that protein may interact with LdPCNA with a consensus PIP box. LdPCNA forms a ring with its trimeric structure. The inner surface of the ring is composed of 12 ␣-helices, whereas the outer surface is composed of 54 ␤-sheets, 3 ␣-helices, and 3 IDCLs. Validation of differential expression of LdPCNA in SAGsensitive LdSS and SAG-resistant LdSR strains of L. donovani. Differential expression levels of LdPCNA in the SAG-sensitive LdSS strain and the SAG-resistant LdSR strain were determined at the mRNA level by real-time PCR and at the protein level by West- FIG 2 (A) SDS-PAGE (8% gel) analysis of glutaraldehyde cross-linked purified LdPCNA. Lane 1, molecular mass markers; lane 2, cross-linked purified trimeric rLdPCNA; lane 3, untreated purified monomeric rLdPCNA. (B) Size exclusion chromatographic profile of rLdPCNA. The size exclusion chromatography was carried out using a Superdex 200 10/300 GL column preequilibrated with buffer (50 mM Tris-HCl [pH 7.2], 200 mM NaCl). The single elution peak of rLdPCNA was observed in the chromatographic profile. Molecular masses of marker proteins are 440 kDa for ferritin, 158 kDa for aldolase, 75 kDa for conalbumin, 43 kDa for ovalbumin, 29 kDa for carbonic anhydrase, and 13.7 kDa for RNase A. June 2014 Volume 58 Number 6 aac.asm.org 3001 Downloaded from http://aac.asm.org/ on October 15, 2017 by guest FIG 1 Cloning, expression, and purification of rPCNA. (A) Specific PCR of PCNA. Lane 1, 1-kb ladder; lanes 2 and 3, amplified PCR product at 882 bp. (B) Clone Tandon et al. Downloaded from http://aac.asm.org/ on October 15, 2017 by guest FIG 3 In silico structure analysis of LdPCNA. (A) Sequence alignment of the template and the protein. Residues highlighted in red correspond to identical/ conserved residues, while residues in red text are similar in the two proteins. (B) Superimposed structure of Leishmania donovani PCNA (cyan) and template 1VYM (magenta). (C) Ribbon representation of LdPCNA as a trimer. Subunit A is colored green, subunit B is colored blue, and subunit C is colored red. (D) Ramachandran plot of the homology-modeled structure of PCNA of L. donovani. The different colored areas indicate “disallowed” (white), “generously allowed” (light yellow), “additional allowed” (yellow), and “most favored” (red) regions. (E) Schematic diagram of L. donovani PCNA modeled protein from the PDBsum. The wiring diagram shows the protein’s secondary structure elements (␣-helices and ␤-sheets) together with various structural motifs such as ␤- and ␥-turns and ␤-hairpins with their corresponding amino acid residues. (F) Superimposition of monomeric LdPCNA (blue) with monomeric E. histolytica PCNA (yellow) and Homo sapiens (green). 3002 aac.asm.org Antimicrobial Agents and Chemotherapy Role of PCNA in SAG Resistance TABLE 2 Fold increase in expression of PCNA at different promastigote and amastigote stages in the SAG-resistant LdSR strain in comparison to the SAG-sensitive LdSS strain of L. donovani as determined by Western blotting and real-time PCR Fold increase (mean ⫾ SD) of PCNA in LdSR strain by: Parasite stage Western blotting Real-time PCR Log promastigote Stationary promastigote Procyclic Metacyclic Amastigote 3.67 ⫾ 0.39 3.29 ⫾ 0.91 3.66 ⫾ 0.88 3.50 ⫾ 0.72 4.98 ⫾ 0.46 3.48 ⫾ 0.77 2.91 ⫾ 0.55 3.14 ⫾ 0.77 2.88 ⫾ 0.96 4.82 ⫾ 0.53 June 2014 Volume 58 Number 6 DISCUSSION Pentavalent antimony has been the most commonly used drug to treat Leishmania patients. However, it has serious side effects and requires a prolonged course of treatment and its use has been stopped due to its loss of efficacy in some regions because of the increasing parasite resistance to the drug. Although newer treatments exist, they are not optimal due to the problems of toxicity, high price, or difficulty in administration (24). Coinfection with HIV poses an additional challenge (25, 26). To understand the mechanism of drug resistance, studies were conducted in the laboratory by generating the antimonial drug-resistant strain through stepwise exposure to antimony. However, in vitro unresponsiveness generated in the laboratory does not necessarily translate to clinical resistance (6, 27). Therefore, information collected from the studies on clinical isolates related to SAG resistance is of paramount importance in revealing the mechanism of FIG 4 Differential expression of PCNA in the SAG-resistant strains LdSR1 and LdSR2 and the SAG-sensitive Dd8 strain of L. donovani. RNA isolated from the late log phase of the different strains was used as a template to prepare cDNA. Differential expression was assessed by real-time PCR. All quantifications were normalized to the housekeeping gene ␣-tubulin. Significance values indicate the difference between the different strains and LdSS (ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001). aac.asm.org 3003 Downloaded from http://aac.asm.org/ on October 15, 2017 by guest ern blot analysis. At the log phase, stationary phase, and isolated procyclic and metacyclic stages of promastigotes, the LdPCNA transcript and protein levels were observed to be ⬃3-fold higher in the LdSR strain than in the LdSS strain. Moreover, this difference in expression was found to be enhanced further to ⬃5-fold at the amastigote stage (Table 2). Expression of LdPCNA was also evaluated in the promastigote stage of other resistant strains (LdSR1 and LdSR2), in which LdPCNA was found to be overexpressed by ⬃2.5- and ⬃2-fold, respectively, compared with that in LdSS (Fig. 4). Overexpression of LdPCNA in SAG-sensitive LdSS strain of L. donovani. To confirm the role of PCNA in SAG resistance, it was overexpressed in the SAG-susceptible L. donovani isolate LdSS and the SAG-resistant clinical isolate LdSR. LdPCNA was expressed as a GFP fusion protein. Its overexpression was validated by Western blotting of the total promastigote lysate probed with either an anti-GFP antibody (Fig. 5A) showing expression of the exogenous protein or an anti-LdPCNA antibody (Fig. 5B) showing both the endogenous and the exogenous proteins simultaneously. The band intensity of the LdPCNA-GFP protein was 1.84fold higher (calculated by densitometric analysis using ImageJ software) in LdSS-PCNA-GFP transfectants maintained at 50 ␮g/ml than that in transfectants maintained at 25 ␮g/ml, suggesting that the copy numbers of the fused protein in transfectants varied with the G418 concentration: the higher the concentration of G418, the higher the expression of the LdPCNA-GFP protein (Fig. 5D). The case with LdSR-PCNA-GFP, wherein the expression of LdPCNA increased with elevated concentrations of G418 in the culture medium, was similar (Fig. 5E). In vitro assay for drug sensitivity. Further, parasites overexpressing LdPCNA were analyzed for their susceptibility to SbV and SbIII. The IC50 (mean ⫾ standard deviation [SD]) of LdSS-PCNAGFP for SbV growing at 25 ␮g/ml was 52.86 ⫾ 1.72 ␮g/ml, ⬃1.3fold higher (P ⬍ 0.01) than that for the wild-type LdSS control (IC50 ⫽ 41.26 ⫾ 0.641 ␮g/ml). Moreover, on increasing the G418 concentration for LdSS-PCNA-GFP to 50 ␮g/ml, the IC50 was increased by ⬃1.6-fold (IC50 ⫽ 66.50 ⫾ 3.93, P ⬍ 0.001) in comparison with that for LdSS, whereas LdSS-GFP control transfectants exhibited similar IC50 at all G418 concentrations. The IC50 of SbV were also significantly increased by 1.21-fold (P ⬍ 0.01) and 1.3-fold (P ⬍ 0.001) against LdSR-PCNA-GFP transfectants maintained in 25 ␮g/ml or 50 ␮g/ml G418, respectively (Table 3). The variations in the IC50 of SbIII for wild-type and transfectants were found to agree with those of SbV. Qualitative analysis of DNA fragmentation in promastigotes after SbIII treatment. DNA fragmentation was readily visible in the case of the SbIII-treated LdSS promastigotes, while LdPCNAoverexpressing SbIII-treated transfectants exhibited lesser fragmentation than LdSS and LdSS-GFP. No fragmentation was detected in the case of the untreated promastigotes (Fig. 6A). Determination of replication inhibition in wild-type/transfectant promastigotes with SbIII treatment. DNA strand synthesis was measured by incorporation of bromodeoxyuridine (BrdU), a thymidine analog. DNA synthesis was decreased after treatment with SbIII at 24 h in all three variants of parasites. However, LdPCNA-overexpressing transfectants exhibited increased DNA replication in comparison with that for wild-type LdSS (P ⬍ 0.01) after treatment with SbIII (Fig. 6B). NO production by macrophages infected with wild-type and transfectant parasites. Significant nitrite production was observed at 24 h and 48 h after treatment with SAG. Macrophages infected with a SAG-sensitive LdSS strain generated maximum levels of nitrite after treatment with SAG which were significantly higher than levels for those infected with a SAG-resistant LdSR strain (P ⬍ 0.001). On the contrary, macrophages infected with the LdPCNA-overexpressing mutant exhibited less NO production upon induction with SAG than those infected with wild-type LdSS and control LdSS-GFP (P ⬍ 0.001) at these time points (Fig. 7). Tandon et al. drug resistance, which is still not very clear. One of the differential proteomics studies that was conducted by Kumar et al. (7) as the first step in resolving the mechanism of drug resistance using clinical isolates identified PCNA as a protein expressed differently in drug-sensitive and -resistant strains. Extensive studies conducted on PCNA and its functions in other organisms in the past few years have revealed that PCNA plays a vital role in several biological processes that appear distinct but have roles in DNA metabolism in common. PCNA is an essential component of the DNA replication machinery, function- TABLE 3 Susceptibility of L. donovani wild-type isolates and transfectants to SbV and SbIIIa Parasite IC50 (␮g/ml) of SbV against intramacrophage amastigotes LdSS 41.2 ⫾ 0.6 LdSS-PCNA-GFP 0 ␮g/ml G418 25 ␮g/ml G418 50 ␮g/ml G418 41.6 ⫾ 0.8 52.8 ⫾ 1.7 66.5 ⫾ 3.9 ⬎0.05 ⬍0.01 ⬍0.001 24.4 ⫾ 0.7 33.3 ⫾ 2.1 43.4 ⫾ 1.8 ⬎0.05 ⬍0.01 ⬍0.001 LdSS-GFP 0 ␮g/ml G418 25 ␮g/ml G418 50 ␮g/ml G418 41.7 ⫾ 0.2 42.1 ⫾ 0.7 43.5 ⫾ 1.3 ⬎0.05 ⬎0.05 ⬎0.05 24.4 ⫾ 0.6 24.9 ⫾ 1.2 24.6 ⫾ 0.6 ⬎0.05 ⬎0.05 ⬎0.05 LdSR 457.8 ⫾ 12.3 LdSR-PCNA-GFP 0 ␮g/ml G418 25 ␮g/ml G418 50 ␮g/ml G418 470.3 ⫾ 16.3 554.2 ⫾ 15.4 580.5 ⫾ 16.6 ⬎0.05 ⬍0.01 ⬍0.001 245.2 ⫾ 11.4 291.3 ⫾ 8.8 313.9 ⫾ 12.6 ⬎0.05 ⬍0.05 ⬍0.01 LdSR-GFP 0 ␮g/ml G418 25 ␮g/ml G418 50 ␮g/ml G418 471.5 ⫾ 13.5 475.2 ⫾ 13.5 479.3 ⫾ 15.5 ⬎0.05 ⬎0.05 ⬎0.05 231.3 ⫾ 11.5 241.4 ⫾ 8.4 248.6 ⫾ 8.8 ⬎0.05 ⬎0.05 ⬎0.05 b P IC50 (␮g/ml) of SbIII against promastigotes Pb 24.0 ⫾ 0.3 237.7 ⫾ 9.4 a Parasite inhibition by SbV in intracellular amastigotes and by SbIII in promastigotes was calculated at drug concentrations of 0, 5, 10, 20, 40, 80, 160, 320, and 640 ␮g/ml. Each value in the table represents the mean ⫾ SD of three separate assays. b The significance values indicate the differences between wild types and their corresponding transfectant parasites at P ⬍ 0.05, P ⬍ 0.01, and P ⬍ 0.001. 3004 aac.asm.org Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on October 15, 2017 by guest FIG 5 Overexpression of PCNA-GFP in the LdSS and LdSR strains of L. donovani. (A) Western blot analysis of the SLD of wild-type LdSS (lane 1), mutant LdSS-GFP (lane 2), and LdSS-PCNA-GFP (lane 3) parasites blot probed with anti-PCNA antibody (Ab). Two bands in lane 3 indicate endogenous (⬃32 kDa) and exogenous (⬃58 kDa) PCNA, respectively. (B) Western blot analysis of the SLD of wild-type LdSS (lane 1), mutant LdSS-GFP (lane 2) and LdSS-PCNA-GFP (lane 3) parasites blot probed with anti-GFP Ab. (C) Images of PCNA-GFP-overexpressing L. donovani transfectants under a fluorescence microscope (left), immunostaining of the cells with 4=,6-diamidino-2-phenylindole (DAPI) (center), and merged image (right). (D) Western blot showing increased expression of the LdPCNA-GFP fused protein with increasing concentrations of G418. Lanes 1, 2, and 3 in panels D and E represent SLD of LdSS-PCNA-GFP and LdSRPCNA-GFP transfectants, respectively, grown at 0, 25, and 50 ␮g/ml of G418 and probed with anti-PCNA Ab. Role of PCNA in SAG Resistance FIG 6 (A) Determination of DNA fragmentation in wild-type and PCNAoverexpressing promastigotes after Sb treatment. Wild-type LdSS and transfectant LdSS-GFP and LdSS-PCNA-GFP (at 50 ␮g/ml G418) promastigotes were treated with 30 ␮g/ml SbIII promastigotes for 24 h. Qualitative analysis of DNA fragmentation was performed by agarose gel electrophoresis of DNA extracted from 1 ⫻ 108 parasites. Lanes 2, 4, and 6 contain 20 ␮g DNA isolated from SbIII-treated LdSS-PCNA-GFP, LdSS, and LdSS-GFP promastigotes, respectively, while lanes 1, 3, and 5 are loaded with DNA of untreated LdSSPCNA-GFP, LdSS, and LdSS-GFP promastigotes, respectively. Lane M represents a 100-bp ladder. (B) Determination of DNA replication inhibition in wild-type/transfectant promastigotes with SbIII treatment by the BrdU assay. Samples (100 ␮l) of wild-type LdSS and transfectants (LdSS-PCNA-GFP and LdSS-GFP growing at 50 ␮g/ml G418 concentration) in complete RPMI 1640 were plated in 96-well plates at 2 ⫻ 106 cells/ml and incubated with 100 ␮l of 30 ␮g/ml of the drug for 24 h, followed by BrdU labeling for 8 h. For a negative control, cells without added BrdU were also included. Newly synthesized DNA was detected using the BrdU cell proliferation assay (Calbiochem), following the manufacturer’s protocol. Absorbance was measured at 450 to 595 nm using a SpectraMax plate reader (Molecular Devices). All samples were analyzed in triplet in three independent experiments. Differences between wildtype and transfectant parasites were considered significant at P ⬍ 0.01 (ⴱⴱ). O.D., optical density. ing as the accessory protein for DNA polymerase ␦ (Pol␦), required for processive chromosomal DNA synthesis, and DNA polymerase ε (Polε). The protein augments considerably the processivity of these enzymes by forming a trimeric ring around DNA, anchoring them to the DNA. PCNA is also required for DNA recombination and repair. In addition, it was shown to interact with cellular proteins involved in cell cycle regulation and checkpoint control (28). Expression and purification of rLdPCNA were previously reported for L. donovani strain S1 (29), but here entirely different conditions for expression and purification of rPCNA of the LdSR strain were optimized, and further characterization studies were conducted using this protein. We observed about a 3- to 4-fold difference in the protein as well as mRNA levels of LdPCNA in all the promastigote stages June 2014 Volume 58 Number 6 after treatment with SAG. Mouse cell line J774A.1(MQ) was infected with the wild-type SAG-sensitive strain LdSS and the SAG-resistant strain LdSR, the PCNA-overexpressing SAG-sensitive LdSS-PCNA-GFP transfectant, and GFP-overexpressing LdSS-GFP transfectant parasites. After 16 h, free promastigotes were washed with serum-free medium and infected macrophages were incubated with medium containing SAG (30 ␮g/ml) at 37°C. A 100-␮l sample of supernatant was collected from each well after 12 h, 24 h, and 48 h and mixed with 100 ␮l of Griess reagent and incubated for 10 min and the optical density of the reaction was taken at 540 nm. Untreated infected macrophages and uninfected macrophages (uninf MQ) served as controls. Differences between groups were considered significant at P ⬍ 0.01 (ⴱⴱ) and P ⬍ 0.001 (ⴱⴱⴱ). (i.e., log, stationary, and isolated procyclic and metacyclic) between the SAG-resistant and SAG-sensitive strains, indicating that the differential expression levels of this protein can be controlled at the DNA level, e.g., for gene duplication. LdPCNA expression was increased further to about 5-fold in the LdSR strain at the pathogenic amastigote stage, demonstrating that this protein might play a positive part in drug resistance. The overexpression of LdPCNA was also checked in other SAG-resistant strains, viz. LdSR1 and LdSR2, and was found to be higher in these strains too. This further strongly supports the view that overexpression of LdPCNA is associated with SAG resistance. Transfection studies using overexpression of a gene have been successfully utilized in several reports to assess the effect on Leishmania phenotypes. Overexpression of some proteins, viz. H2A, HSP-83, and P299, in sensitive L. donovani isolates has resulted in increased resistance to the antimonies (30–32). In our study also, we showed that overexpression of the LdPCNA protein decreased the susceptibility of the sensitive parasite to antimonials. Moreover, with the increasing overexpression of LdPCNA-GFP by elevating G418 concentrations, a decline in antimony susceptibility was observed in both promastigotes and intracellular amastigotes. This reveals a direct correlation of PCNA protein concentrations with antimony resistance in Leishmania. Although several proteins along with PCNA were identified as being overexpressed in the SAG-resistant clinical isolate through differential proteomics, viz. ABC transporter, HSP-83, GPI protein transamidase, cysteine-leucine-rich protein, 60S ribosomal protein L23a, proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, and tubulin-beta chain (7), each has a different distinct role in cellular aac.asm.org 3005 Downloaded from http://aac.asm.org/ on October 15, 2017 by guest FIG 7 Estimation of nitric oxide production by the infected macrophages Tandon et al. 3006 aac.asm.org development of LdPCNA-binding peptides containing the PIP box. Multiple proteins can interact with LdPCNA, mainly through its IDCL with a consensus motif, present in the interacting proteins and named the LdPCNA-interacting protein box (PIP box), which has a QXX(M/L/I)XX(F/Y)(F/Y) consensus sequence (40). Many proteins have a PIP box, which is a physiologically relevant interaction site for PCNA. Rational designing of peptides having PIP box sequences and using them as competitive inhibitors to disrupt the vital protein-PCNA interactions have provided excellent leads for antiproliferative therapy in cancers (41). A number of studies using peptides as antileishmanials have been carried out or are underway (42, 43). LdPCNA and HuPCNA have only 39% homology; therefore, designing of peptides with PIP boxes which could specifically bind to LdPCNA could pave the way to novel antileishmanials. Moreover, as PCNA has been found to be overexpressed in different SAG-resistant isolates, its detection could be used as a marker in SAG resistance. ACKNOWLEDGMENTS We are grateful to the director of the CDRI/CSIR, Lucknow, for providing the facilities to make this study possible. This work was supported by a grant from the CSIR Supra Institutional Project (SIP0026). Financial assistance to R.T., R.K.B., and P.M. from CSIR, New Delhi, and to S.C., A.K., and S.D. from UGC, New Delhi, is gratefully acknowledged. We declare no conflicts of interest. REFERENCES 1. Alvar J, Yactayo S, Bern C. 2006. Leishmaniasis and poverty. Trends Parasitol. 22:552–557. http://dx.doi.org/10.1016/j.pt.2006.09.004. 2. Herwaldt BL. 1999. Leishmaniasis. Lancet 354:1191–1199. http://dx.doi .org/10.1016/S0140-6736(98)10178-2. 3. Sundar S, Chakravarty J, Rai VK, Agrawal N, Singh SP, Chauhan V, Murray HW. 2007. Amphotericin B treatment for Indian visceral leishmaniasis: response to 15 daily versus alternate-day infusions. Clin. Infect. Dis. 45:556 –561. http://dx.doi.org/10.1086/520665. 4. Ouellette M, Drummelsmith J, Papadopoulou B. 2004. Leishmaniasis: drugs in the clinic, resistance and new developments. Drug Resist. Updat. 7:257–266. http://dx.doi.org/10.1016/j.drup.2004.07.002. 5. Croft SL, Sundar S, Fairlamb AH. 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19:111–126. http://dx.doi.org/10.1128/CMR.19.1 .111-126.2006. 6. Ashutosh Sundar S, Goyal N. 2007. Molecular mechanisms of antimony resistance in Leishmania. J. Med. Microbiol. 56:143–153. http://dx.doi.org /10.1099/jmm.0.46841-0. 7. Kumar A, Sisodia B, Misra P, Sundar S, Shasany AK, Dube A. 2010. Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani. Br. J. Clin. Pharmacol. 70:609 – 617. http://dx.doi .org/10.1111/j.1365-2125.2010.03716.x. 8. Dube A, Singh N, Sundar S. 2005. Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persist in in vitro and in vivo experimental models. Parasitol. Res. 96:216 –223. http://dx.doi .org/10.1007/s00436-005-1339-1. 9. Lakshmi V, Pandey K, Kapil A, Singh N, Samant M, Dube A. 2007. In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani. Phytomedicine 14:36 – 42. http: //dx.doi.org/10.1016/j.phymed.2005.10.002.. 10. Da Silva R, Sacks DL. 1987. Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation. Infect. Immun. 55: 2802–2806. 11. Chang KP. 1980. Human cutaneous Leishmania in a mouse macrophage line: propagation and isolation of intracellular parasites. Science 209: 1240 –1242. http://dx.doi.org/10.1126/science.7403880. 12. Gupta SK, Sisodia BS, Sinha S, Hajela K, Naik S, Shasany AK, Dube A. 2007. Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania don- Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on October 15, 2017 by guest metabolism and may be directly or indirectly associated or involved in the SAG resistance mechanism. One of these proteins, 60S ribosomal protein L23a, has been found to be associated with the cellular proliferation in SAG-resistant parasites (33). Besides these proteins, several other overexpressed or downregulated proteins, e.g., mitogen-activated protein (MAP) kinase 1, histone 2A, etc. as studied elsewhere, have been reported to play an important role in SAG resistance. Whereas the downregulation of MAP kinase 1 of L. donovani has been demonstrated to modulate SAG resistance in clinical isolates (34), overexpression of H2A, a component of histone octamer, which facilitates the formation of higher-order chromatin structures, resulted in conversion of SAG-sensitive Leishmania parasites into a resistant phenotype (30). As these identified proteins perform diversified cellular functions, the mechanism of SAG resistance seems to be a complex mechanism involving several factors and pathways. Therefore, it is possible that drastic changes in the sensitivity of the parasite toward the drug may not be achieved by mere modulation of a single factor or protein. This possibility probably could be the basis behind the minor but statistically significant increase in the IC50 of SAG (1.6-fold) in PCNA-overexpressing SAG-sensitive parasites. Moreover, overexpression of PCNA also increased SAG resistance in the LdSR-resistant phenotype, which also indicates the involvement of PCNA in SAG resistance. Further, in order to assess whether the changes in the drug susceptibility are only due to overexpression of the protein or also due to alterations in the protein activity, the sequencing of the PCNA gene of both of the LdSR and LdSS isolates was carried out and revealed no changes in the nucleotide and amino acid sequences (data not shown). This observation negates the possibility of any changed activity of the protein due to the different amino acid sequences in the two clinical isolates. Furthermore, this also strengthens the possibility that overproduction of the protein and not its altered activity is responsible for increased SAG resistance in the parasites. In order to reveal the possible role of PCNA in the mechanism of SAG resistance, further experiments were carried out. It is well documented that the potential molecular targets of SbIII are associated with DNA replication, structure, and repair (35, 36). Previously, SbIII had been reported to induce DNA fragmentation in Leishmania, which prevents further replication and cell proliferation and ultimately causes cell death. In our study we have shown that overexpression of LdPCNA resulted in lesser DNA fragmentation. Moreover, after SbIII treatment, LdPCNA-overexpressing transfectants exhibited more DNA replication and cell proliferation than the wild type, which indicates that overexpression of PCNA has protected against the DNA damage induced by antimonials. Further detailed study is required to understand its biochemical and molecular mechanisms. Previously, it was reported that SAG treatment alone induced both reactive oxygen species (ROS) and NO in L. donovani-infected murine macrophages (37–39), which was observed in the present study wherein the overexpression of LdPCNA inhibited SAG-induced NO production in the infected macrophages. Further studies might provide insight into whether LdPCNA is directly or indirectly involved in the alteration of the downstream signaling pathway of NO-induced killing of Leishmania. The present study divulges the important role of PCNA in preventing DNA damage induced by antimonials. Development of novel drugs targeting LdPCNA might therefore be useful, especially against SAG-resistant parasites. One such approach is the Role of PCNA in SAG Resistance 13. 14. 15. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 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