previously [38]. To generate double knockout parasites, second allelic replacement construct was transfected into the HKO parasites. Proper insertion of cassettes was confirmed by PCR to detect the presence of replacement constructs into the transfor- mant genomic DNA. Primers were also designed using the intergenic sequence upstream of CYP5122A1 gene, the antibiotic resistance marker ORFs and the CYP5122A1 gene, such that amplification using these primers of expected molecular weight would only occur upon insertion at the correct locus. The leishmanial expression vector pXG-GFP +2 (a kind gift from Dr. S. Beverley) containing the CYP5122A1 gene was used for complementation of CYP5122A1 through episomal expression in the HKO parasites. Complementation was confirmed by GFP expression and detection of fusion m-RNA and GFP-fusion protein in the transformants. Immunocytochemistry and growth curve Fixed (2% formaldehyde), digitonin or saponin (0.01%) permeabilized cells were stained using rabbit anti-CYP5122A1 antibody (1:500) followed by labelling with Alexa Fluor 488 conjugated secondary antibody (1:750). For co-localization, MitoTrackerH Red (0.5 mM/106 cells) was used to visualize mitochondria while ER-TrackerTM Blue-White DPX (1 mM/106 cells), anti-BIP antibody (1:1000) were used to identify the endoplasmic reticulum (ER). Anti-GAPDH antibody (1:50) was used to identify glycosomes. Stained cells were analysed with a Nikon Eclipse TE2000E microscope (Nikon, Japan). FACS Calibur (Becton Dickinson, NJ) equipped with a 488 nm air cooled argon ion laser was used for flow cytometric analysis as described previously [45]. Mean fluorescence intensities per unit area of WT and HKO cells stained with anti-CYP5122A1 antibody were measured using the software ImagePro Plus (Media Cybernetics). For growth curve, equal number of cells (107 or 56106) was plated and cell numbers analysed by haemocytometer based counting every 24 h. Measurement of DYm, Ca2+, ROS and ATP Ca2+ and DYm was measured using Rhod-2 AM and JC-1 as probe as described previously [6]. For measurement of ROS, the cells (107) were loaded with the cell permeant probe CM- H2DCFDA (2 mg/mL) as described previously [6] and fluores- cence measurements were taken at 507/530 nm using a FluoStar Optima fluorescence reader (BMG Labtechnologies Inc., Ger- many). ATP was measured in a reaction buffer containing 1 mM dithiothreitol, 0.5 mM luciferin and 12.5 mg/mL luciferase using a Fluostar Omega multi-label detection system as described previously [6]. Infection In vitro Leishmania-macrophage infection was carried out as described previously [7] at a parasite: macrophage ratio of 10:1. For in vivo infection, Syrian golden hamsters were injected intra- cardially with 108 parasites in stationary phase of growth. After 8 weeks, the animals were euthanized and spleen slices were used to prepare splenic smears. Smears were stained with Giemsa stain and scored for infected macrophages. Estimation of ergosterol Sterols were isolated as described by Arthington-Skaggs et al. [46], with slight modifications. Briefly, cells were suspended in 25% alcoholic KOH and incubated at 85uC (1 h) followed by cooling to room temp and addition of sterile distilled water and n- heptane mixture. Standard ergosterol dilutions and sterol CYP5122A1, a Novel Cytochrome P450 of Leishmania extraction samples from WT, HKO and complemented strain of parasites were spotted on a Silica Gel G plate, resolved using a binary solvent (hexane/ethyl acetate (75/25)) and visualized using Mo’s stain (12.5 g of ammonium molybdate (VI) tetrahydrate, 5.0 g of ammonium cerium (IV) sulphate, 50 ml of concentrated sulfuric acid volume raised to 500 mL with water). For GC-MS, the sterol extracts were dried under nitrogen gas, resuspended in n- hexane and derivatized with BSTFA containing 1% TMCS at 70uC for 1 h. The derivative mixture was dried under nitrogen to remove excess BSTFA and subsequently re-dissolved in n-hexane. Agilent 7890A gas chromatography instrument coupled to an Agilent 5975C mass spectrometer and an Agilent ChemStation software (version G1701EA, Agilent Technologies, Palo Alto, CA) was used for GC-MS. For separation, a HP-5MS capillary column (30 m60.25 mm i.d) was used with the column temperature was set at 100uC (held for 5 min for injection) programmed at 20uC min21 to 200uC (held for 10 min, followed by 10uC min21 to 230uC) and finally at 5uC min21 to 320uC (5 min). Injection temperature was set at 260uC. High purity helium was used as carrier gas of 1.0 mL min21 flow-rate. The spectrophotometers were operated in electron-impact (EI) mode, full scan of 40– 550 amu or selected ion monitoring (SIM) was used, the ionization energy was 70 eV. Calibration curve was generated using n- hexane stock solutions of standard ergosterol dilutions (5- 500 ng/ mL) in duplicates and plotting the peak area versus the concentration. Statistical analysis Data were analysed by student’s unpaired t-test. Values were considered significantly different at P#0.05. All experiments were repeated 3–4 times. Data are expressed as the mean 6 SE of several independent experiments. Supporting Information Figure S1 Phylogenetic analysis of CYP5122A1. A phylo- gram generated from CYP450-like sequences analysed by ClustalW showing distances between CYP450-like proteins of various species. Scale represents 0.06 nucleotide substitutions per site. (TIF) Figure S2 Predicted secondary structure. Schematic representation of the predicted secondary structures in and around the active site generated using the PSIPRED server hosted at the Bloomsburry Centre for Bioinformatics (University College, London and Birkbeck college). PTG, Proton transfer groove; SC, stabilization core; HBL, Heme binding loop are highlighted in yellow. Active site cysteine indicated by arrow. (TIF) Figure S3 Ramachandran plot of CYP5122A1. Ramachan- dran plot generated for CYP5122A1 protein structure modelled using SWISS-MODEL shows majority of the residues to fall into the favourable region of the plot. (TIF) Figure S4 Analysis of pBSK+CYP5122A1-Hyg HKO. A: (i) Insertion of allelic replacement construct into the genomic DNA was confirmed by PCR for hygromycin and knockout-hygromycin construct using primers that span the 59 homologous sequence (Forward primer) and the hygromycin gene (Reverse primer). Lane 1: fragment amplified for the hygromycin resistance gene; lane 2: 1 kb DNA ladder; lane 3: fragment amplified for the knock out hygromycin construct. (ii) Confirmation of insertion of the replacement construct at the correct locus was assessed by PCR. PLoS ONE | www.plosone.org 14 September 2011 | Volume 6 | Issue 9 | e25273