Figure 1. Structural analysis of CYP5122A1. A: Protein sequence based schematic representation of CYP450 conserved motifs in CYP5122A1 sequence. TM, Trans-membrane domain; PTG, Proton transfer groove; SC, Stabilization core; HBL, Heme binding loop. B: A ribbon representation of folded CYP5122A1 protein structure generated using SWISS-MODEL server. a-helix, coiled ribbon; b-sheet, arrow; random coil and loop, line. The motifs shown are, stabilization of the core (pink) in the K helix; the proton transfer groove in central part of the I-helix, mauve; heme binding loop, purple; conserved Cys (523), indicated by an arrow. doi:10.1371/journal.pone.0025273.g001 addition, although less predominant, co-localization of CYP5122A1 staining was also seen in the mitochondria identified using MitoTracker RedH or glycosomes marked with glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) (Fig. 3B, a–e, Mito- Tracker red and GAPDH). Therefore, these observations show that CYP5122A1 distribution is confined predominantly to the endoplasmic reticulum and in smaller amounts to the mitochon- dria and the glycosomes. The metacyclic forms of the parasite were isolated and stained with anti-CYP5122A1 antibody and anti-BIP antibody to see if the staining pattern was similar to log phase promastigotes. Co-localization of CYP5122A1 with BIP was observed indicating that there was no apparent difference in the localization of the protein as cells differentiated into metacyclics (Fig. S7). CYP5122A1 is essential for optimal growth of the parasite To determine the importance of CYP5122A1 in the survival of Leishmania donovani, the CYP5122A1 encoding sequence was disrupted by ORFs encoding for neomycin or hygromycin resistance. Gene replacement vectors (pBSK+CYP5122A1Neo or pBSK+CYP5122A1Hyg) were constructed such that ORFs encod- ing neomycin/hygromycin resistance were flanked by sequences homologous to 59 and 39 region of the target gene. These vectors CYP5122A1, a Novel Cytochrome P450 of Leishmania were used to transform wild-type (WT) cells where homologous recombination resulted in one allele being replaced by the antibiotic resistance gene and the resultant parasites had only one functional allele of CYP5122A1 (half knockout, HKO). These parasites with one allele of CYP5122A1 were re-transfected with the vectors containing the second antibiotic resistance gene to obtain replacement of the remaining allele so that parasites expressing no CYP5122A1 could be obtained. These double knockout parasites did not survive in culture suggesting that the gene may be essential for parasite survival. Therefore, we used the HKOs containing one allele of CYP5122A1 for further functional experiments. For characterization of these HKO parasites, the successful deletion one WT copy of the CYP5122A1 gene was confirmed by PCR with genomic DNA of the HKOs using primers (primer positions marked in the schematic, Fig. 4A) for neomycin (Fig. 4B, lane 2), as well as a forward primer spanning the 59 end of the CYP5122A1 gene and reverse primer for the neomycin (Fig. 4B, lane 5) or hygromycin ORF (Fig. S4A (i), lane 1) and 59-hygromycin fragment (Fig. S4A (i), lane 3). This confirmed the presence of the neomycin/hygromycin resistance gene. To confirm the correct targeting of the replacement construct to the CYP5122A1 locus, a forward primer was designed from the intergenic region upstream of CYP5122A1 gene that has not been included in the replacement construct (Primer F2 59- GTTGCGACTTACCTTCTACGTGTG-39; Fig. 4A). Two re- verse primers were designed, one for each allele of the half knock out. The first reverse primer corresponded to sequences in the antibiotic resistance marker neomycin (Neo Internal Primer, 59- CAAGGTGAGATGACAGGAGATC-39; Fig. 4A) or the hygro- mycin resistance marker (Hyg Internal Primer, 59-CAAGCAC TTCCGGAATCGGGC-39) used to replace the CYP5122A1 gene and would identify the allele that has undergone homologous recombination. To identify the other allele, we designed a reverse primer (Primer P2 59-CCTTCTTCCACTGCTCATCC-39; Fig. 4A) in the region of CYP5122A1 gene that has been replaced on the corresponding allele and hence would only amplify fragments from the intact allele. We therefore expected amplifica- tion with primer pair F2/P2 only (Fig. 4C, lane 1) in the WT while the HKO parasites would give positive PCR reactivity with both F2/P2 as well as F2/Neo Int (Fig. 4C, lanes 3 and 4) or F2/Hyg Int (Fig. S4A (ii)). To check the functional output of knockout of one allele, cell extracts from WT and HKO parasites were probed with anti- CYP5122A1 antibody to detect the extent of CYP5122A1 expression. Significantly lower amount of CYP5122A1 was expressed in the HKOs where replacement was made with the neomycin cassette (Fig. 4D, lane 1, Fig. 4E) and the hygromycin cassette (Fig. S4B, lane 2; S4C) as compared to WTs in respective blots. This knockdown was specific for CYP5122A1 because HKO parasites did not show any down-regulation of CYP710C1, another CYP450 gene of Leishmania donovani that was used as a CYP450 control (Fig. S4D). Protein expression was further confirmed by comparative fluorescence microscopy where staining intensity for CYP5122A1 was lower in HKO cells (Fig. 4F, c–d) as compared to WTs (Fig. 4F, a–b). To rule out the possibility of apparent higher staining due to larger size of the cell, mean fluorescence intensity per unit area per cell was measured microscopically. Fig. 4G shows the mean fluorescence intensity per unit area of the stained cells where the amount of CYP5122A1 staining in the HKOs/unit area was significantly less as compared to the WTs. To assess if the reduced expression of CYP5122A1 compro- mised cellular physiology, various parameters were checked in the HKO parasites. Morphologically, the HKOs were smaller in size than their WT counterparts at all stages of the in vitro life cycle (Fig. 5A). The division rate of the HKO cells was substantially PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25273