Lane 1: amplicons generated from HKO genomic DNA as template with primers F2/P2 indicating the presence of an intact CYP5122A1 allele (primer positions indicated in schematic in Fig. 4A); Lane 2: amplicons generated from HKO-Hyg genomic DNA as template with primers F2/HI indicating the presence of insertion of hygromycin resistance ORF at the correct locus. B: Western blot analysis of cell lysates from WT and HKO parasites with anti-CYP5122A1 antibody showing the decreased level of CYP5122A1protein in the HKOs. Loading was normalized with Tubulin–a (50 kDa). C: Bar graph representing averaged densitometric analysis of immunoblots represented in B, mean6 SE, n = 3. D: Western blot analysis of cell lysates from WT and HKO parasites using anti-CYP710C1 antibody showing equal levels of the CYP710C in the WT and the HKO parasites. Loading was normalized with tubulin-a (50 kDa). E: Comparison of growth and survival of WT and HKO parasites in vitro over a period of 6 days in culture. Mean 6 SE, n = 3. F: Bar graph showing percentage viability of WT and HKO parasites in response to treatment with PAT. Note the increased susceptibility of HKOs as compared with WT. Mean 6 SE, n = 4; * P#0.05. G: Photomicrographs of macrophage infected with WT and HKO parasites stained with Syto 11 Green fluorescent nucleic acid stain. a: nomarski image; b: Syto 11 Green stained image; c: merge of a and b. Scale, 5 mm. (TIF) Figure S5 A: Bar graph shows percentage viability of WT and HKO parasites in response to treatment with H2O2. Mean 6 SE, n = 4; * P#0.05. B: Photomicrographs of J774A.1 macrophages infected with WT and HKO L. donovani parasites at multiplicity of infection at 1:25 and 1:50, stained with Syto 11 Green. Scale, 20 mm. (TIF) Figure S6 Effects of complementation of CYP5122A1 in HKOs. A: Agarose gel (1.5%) showing expression of GFP- CYP5122A1 fusion mRNA (GFP Forward primer & CYP5122A1 internal primer) in 2 clonally selected strains of CYP5122A1 complemented parasites. Lane 1: negative control; lane 2 & 3: References 1. Croft SL, Seifert K, Yardley V (2006) Current scenario of drug development for leishmaniasis. Indian J Med Res 123: 399–410. 2. Alvar J, Aparicio P, Aseffa A, Den BM, Canavate C, Dedet JP, et al. (2008) The relationship between leishmaniasis and AIDS: the second 10 years. Clin Microbiol Rev 21: 334–59. 3. Ouellette M, Drummelsmith J, Papadopoulou B (2004) Leishmaniasis: drugs in the clinic, resistance and new developments. Drug Resist Updat 7: 257– 266. 4. Krauth-Siegel RL, Meiering SK, Schmidt H (2003) The parasite-specific trypanothione metabolism of Trypanosoma and Leishmania. Biol Chem 384: 539–549. 5. Mehta A, Shaha C (2004) Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J Biol Chem 279: 11798–11813. 6. Mukherjee SB, Das M, Sudhandiran G, Shaha C (2002) Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. J Biol Chem 277: 24717–24727. 7. Sudhandiran G, Shaha C (2003) Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J Biol Chem 278: 25120–25132. 8. Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124: 128–145. 9. Roberts CW, McLeod R, Rice DW, Ginger M, Chance ML, et al. (2003) Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol Biochem Parasitol 126: 129–142. 10. Urbina JA (1997) Lipid biosynthesis pathways as chemotherapeutic targets in kinetoplastid parasites. Parasitology 114 Suppl: S91–S99. 11. Berger BJ, Fairlamb AH (1993) Cytochrome P450 in trypanosomatids. Biochem Pharmacol 46: 149–157. CYP5122A1, a Novel Cytochrome P450 of Leishmania fusion product amplified from c-DNA prepared from comple- mented parasites; lane 4: 1 kb DNA Ladder; lane 5: Positive control amplified using pXG-GFP+2CYP5122A1 plasmid as the template. B: Western blot analysis of lysates of HKO and CMPL cells probed for the fusion protein show the presence of GFP- CYP5122A1 protein at expected molecular weight of approxi- mately 96KDa in lysates of complemented parasites. C: Flow cytometric analysis of complemented parasites. D. Photomicro- graphs of HKO (a) and complemented (b) parasites. Scale, 5 mm. E: Bar graph showing percentage viability of WT, HKO and complemented parasites in response to treatment with miltefosine and PAT. Mean 6 SE, n = 4; * P#0.05. F: Photomicrographs of CMPL promastigotes stained with anti-CYP5122A1 antibody and ER-specific markers, ER Tracker Blue-White DPX. Scale, 5 mm. (TIF) Figure S7 Subcellular localization of CYP5122A1 in metacyclic parasites Photomicrographs of Leishmania donovani metacyclic parasites stained with anti-CYP5122A1 antibody and ER-specific marker, BIP. Scale represents 10 mm. (TIF) Acknowledgments The pXG–GFP+2 and the pXG-HYG vector was a kind gift from Dr. Stephen M. Beverley, Department of Molecular Microbiology, Washing- ton University School of Medicine, St Louis, MO. Anti-BIP antibody was a kind gift from Dr. J. D. Bangs, University of Wisconsin, Madison, WI. The assistance of Dr. Gopalan Sampathkumar and Ms. Kavita Aggarwal for Thin Layer chromatography is greatly appreciated. Technical assistance from Mr. G.S. Neelaram is acknowledged. We acknowledge the help of Ms. Archana Ranjan for GCMS and Mr. Nikhil Damle for help in bioinformatic analysis. Author Contributions Conceived and designed the experiments: CS SV. Performed the experiments: SV AM. Analyzed the data: CS SV. Contributed reagents/ materials/analysis tools: CS. Wrote the paper: CS. 12. McLean KJ, Carroll P, Lewis DG, Dunford AJ, Seward HE, et al. (2008) Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv. J Biol Chem 283: 33406–33416. 13. Panwar SL, Krishnamurthy S, Gupta V, Alarco AM, Raymond M, et al. (2001) CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans. Yeast 18: 1117–29. 14. Brumfield KM, Moroney JV, Moore TS, Simms TA, Donze D (2010) Functional characterization of the Chlamydomonas reinhardtii ERG3 ortholog, a gene involved in the biosynthesis of ergosterol. PLoS One 5: e8659. 15. Galea AM, Brown AJ (2009) Special relationship between sterols and oxygen: were sterols an adaptation to aerobic life? Free Radic Biol Med 47: 880–889. 16. de Souza W, Rodrigues JCF (2009) Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs. Interdisciplinary Perspectives on Infectious Diseases 2009: 1–19. 17. Ouellette M, Drummelsmith J, Phillippe, Leprohorn P, Fadili KE, et al. (2008) In. Drug resistance in Leishmania. Leishmania after the genome. Myler P, Fasel N, eds. Norfolk, UK: Caister Academic Press. pp 159–176. 18. Mbongo N, Loiseau PM, Billion MA, Robert-Gero M (1998) Mechanism of amphotericin B resistance in Leishmania donovani promastigotes. Antimicrob Agents Chemother 42: 352–357. 19. Nelson DR (2009) The cytochrome p450 homepage. Hum Genomics 4: 59–65. 20. Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1: REVIEWS3003. 21. Sacks DL, Hieny S, Sher A (1985) Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes. J Immunol 135: 564–569. 22. Gunter TE, Yule DI, Gunter KK, Eliseev RA, Salter JD (2004) Calcium and mitochondria. FEBS Lett 567: 96–102. 23. Sundar S, Chatterjee M (2006) Visceral leishmaniasis - current therapeutic modalities. Indian J Med Res 123: 345–352. PLoS ONE | www.plosone.org 15 September 2011 | Volume 6 | Issue 9 | e25273