data presented here strongly suggests that CYP5122A1 could be a possible drug target as it is functionally important for parasite survival and does not share identity with host CYP450s making it amenable to interception. Moreover, this protein could play an important role in resistance to drugs that use sterols to exert their effects, for which the levels of this protein in drug resistant isolates from endemic areas needs to be investigated. Materials and Methods Reagents ERTrackerTM Blue-White DPX, MitoTrackerH Red, Rhod – 2AM, JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazo- lylcarbocyanineiodide), CM-H2DCFDA, SYTO 11 Green, ATP determination kit and pluronic acid were obtained from Molecular Probes (Eugene, OR). pGEM-TEasy vector system was procured from Promega (Madison, WI). Trizol, First strand c-DNA synthesis kit and Hi-fidelity Taq polymerase were from Invitrogen (Carlsbad, CA). Restriction enzymes, dNTPS, Quick ligase, T4 DNA ligase, and Taq polymerase were from New England BioLabs (Beverley, MA). Miltefosine was purchased from Cayman Chemicals (Ann Arbor, MI). Potassium antimony tartrate (PAT), Dulbecco’s Modified Eagles Medium (DMEM), ergosterol, peanut agglutinin, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide), triton-X-114, amphotericin B, BSTFA (N,O-bis (trimethylsilyl) trifluoroacetamide), TMCS (trimethylchlorosilane), foetal bovine serum and genomic DNA isolation kit were obtained from Sigma Aldrich. Western blotting reagents and enhanced chemiluminescence kit were from Amersham Life Sciences (Piscataway, NJ). CB-XTM Protein assay kit was obtained from G-Biosciences (Maryland Heights, MO). Silica G plates were from Analtech Inc. (Newark, DE). Peptides were commercially synthesized by USV Peptides Ltd. (Bangalore, India). All other chemicals, unless otherwise specified, were from Sigma Aldrich. Antibodies Anti-CYP5122A1 antibody was raised in rabbits using a peptide specific for the CYP5122A1 protein (Gly-Pro-Arg-Gly-Val-Pro- Ser-Val-Asn-Asp-Val-Arg-Asp-Leu). This stretch was selected for raising antibodies for its high antigenicity, hydrophobicity, the presence of coils in the secondary structure and sequence specificity for CYP5122A1 distinct from the other CYP450s annotated in the Leishmania spp. database. Similarly antibody was also raised against another CYP450 of L. donovani, CYP710C1 using the peptide (Cys-Asp-Glu-Lys-Thr-Val-Val-Pro-Lys-Gly- Met-Ile). Rabbit anti-tubulin-a antibody was obtained from Neomarker (Fremont, CA). Anti-BIP antibody was a kind gift from Dr. J. D. Bangs, University of Wisconsin (Madison, WI). Secondary antibodies conjugated to Alexa Flour 488 and 594 and horse radish peroxidase were obtained from Invitrogen (Carlsbad, CA) and Jackson Immunoresearch Laboratories (West Grove, PA) respectively. Anti-GAPDH was from Santa-Cruz Biotechnology (Santa Cruz, CA). Animals Syrian hamsters (Mesocricetus auratus), 3–6 weeks old, were used for in vivo infection. Ethics statement All procedures in this study were carried out as per the guidelines laid down by the ‘‘Institutional Animal Ethics Committee’’ of the National Institute of Immunology, New Delhi. The protocol for this specific study was approved by the same committee via permit no. IAEC/168/07. This study was also CYP5122A1, a Novel Cytochrome P450 of Leishmania approved by the ‘‘Institutional Biosafety Committee’’ via permit no. IBSC110#07. Cells, treatments, extraction and MTT assay Leishmania donovani promastigotes, amastigotes and J774A.1 murine macrophages (ATCC No. TIB-67) were cultured as described earlier [6,7,38]. Metacyclics were isolated by incubating 107 cells in 100 mg/mL peanut agglutinin (PNA) and cells non- adhered to PNA (metacyclics) were separated by centrifugation [21]. To study effects of drugs, parasites in the logarithmic phase were treated with different doses of miltefosine (10 mM to 40 mM), potassium antimony tartrate (100–300 mg/mL) and amphotericin B (1–2.5 mg/mL). For extraction of membrane proteins, cells were resuspended in 0.5% Triton X-114, homogenized and incubated on ice for 90 mins. The insoluble material was removed by centrifugation and the supernatant was incubated at 37uC for one hour following which the aqueous and detergent phases were separated. Ergosterol was added to culture medium at a final concentration of 3 mg/mL. To estimate cytotoxicity, MTT assay was carried out as described previously [38]. Cell lysates, SDS-PAGE and western blotting Cell lysis was performed in a lysis buffer (0.125 M Tris HCl, (pH 6.8) 4% SDS, 20% v/v glycerol, 10% b-mercaptoethanol) and protein content was quantitated using CB-XTM Protein Assay Kit. SDS PAGE, western blots and densitometry were carried out as described previously [6,38]. For western blots, antibodies were used at various dilutions; anti-CYP5122A1 antibody (1:20,000), anti-tubulin-a antibody (1:10,000), secondary antibodies (1:20,000 dilutions). Reactivity was visualized by enhanced chemilumines- cence. Cloning, sequencing and analysis Overlapping sets of primers for the CYP450 protein genes were designed from sequence available with the Leishmania major genome database (LmjF27.0090, Gene DB hosted by the Wellcome Trust Sanger Institute, Hinxton, UK). Using L. donovani genomic DNA as template, an insert was amplified with relevant primers and cloned into pGEM-TEasy vector. Sequencing was carried out by the di-deoxy method [39], at sequencing facility of Delhi University (New Delhi, India). The sequence was submitted to the CYP450 Nomenclature Committee [19] (http://drnelson. uthsc.edu/cytochromep450.html) for assignment. Alignment of the L. donovani sequence with other CYP450s was performed using ClustalW hosted at the European Bioinformatics Institute [40]. The protein sequence derived was subjected to secondary structure prediction program PSIPRED hosted at the Bloomsburry Center for Bioinformatics [41]. The derived protein sequence of CYP5122A1 was also submitted to the SWISS-MODEL homol- ogy modelling server hosted at the Swiss Institute of Bioinformatics [42]. The model generated was viewed in Pymol molecular graphic software (educational use). The stereochemical qualities of this 3D model were checked using Ramachandran plot [43] generated by PROCHECK (EMBL-EBI, UK) (http://www.ebi. ac.uk/thornton-srv/software/ PROCHECK/). WHATCHEK (http://www.cmbi.kun.nl/gv/whatcheck/) server was used to assess the packing quality of the protein model [44]. For CYP5122A1 allelic replacement constructs, ORFs encoding neomycin and hygromycin resistance were inserted between 0.4 kb of 59 and 0.37 kb of 39 of CYP5122A1 homologous regions in the pBlueScript SK+ vector to generate the constructs pBSK+CYP5122A1Neo and pBSK+CYP5122A1Hyg respective- ly. Half knockouts (HKOs) were generated with these constructs through transfection of log phase promastigotes as described PLoS ONE | www.plosone.org 13 September 2011 | Volume 6 | Issue 9 | e25273