CYP5122A1, a Novel Cytochrome P450 Is Essential for Survival of Leishmania donovani Smriti Verma, Ashish Mehta¤, Chandrima Shaha* Cell Death and Differentiation Research Laboratory, Centre for Molecular Medicine National Institute of Immunology, New Delhi, India Abstract Background: Cytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive. Methodology/Principal Findings: A CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca2+ and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol levels by 1.8 times. Phenotype reversal included restored growth pattern and lesser drug susceptibility. Conclusions/Significance: In summary, this study establishes CYP5122A1 as an important molecule linked to processes like cell growth, infection and ergosterol biosynthesis in Leishmania donovani. Citation: Verma S, Mehta A, Shaha C (2011) CYP5122A1, a Novel Cytochrome P450 Is Essential for Survival of Leishmania donovani. PLoS ONE 6(9): e25273. doi:10.1371/journal.pone.0025273 Editor: Mauricio Martins Rodrigues, Federal University of Sa˜o Paulo, Brazil Received April 30, 2011; Accepted August 30, 2011; Published September 23, 2011 Copyright: 2011 Verma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Department of Biotechnology, New Delhi, India (http://dbtindia.nic.in/index.asp) to the National Institute of Immunology (Grant No. BT/03/033/88) and Centre for Molecular Medicine, New Delhi (Grant No. BT/PR/14549/MED/14/1291). The author SV was supported by a fellowship from the Council of Scientific and Industrial Research (CSIR), India (Award No. 9/485(142)/05-EMR-I). The author AM was supported by fellowship from Life Science Research Board, New Delhi (Grant No. DLS/81/48222/LSRB-169/ID/2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cshaha@nii.ac.in ¤ Current address: Research and Development Unit, National Heart Centre, Singapore, Singapore Introduction For survival within host cells, parasites are required to eliminate host generated oxidative stress products, maintain biosynthesis of essential molecules and resist lethal drugs. Leishmania donovani, a kinetoplastid parasite, causes visceral leishmaniasis (VL) in humans and the disease remains a significant problem in the tropics due to emergence of drug resistance [1]. This situation is compounded by overlap of endemic regions of VL with regions of HIV infection where the opportunistic pathogen Leishmania presents a major problem [2]. Leishmania parasites have a digenetic life cycle where infectious metacyclic promastigotes differentiate within the sand fly and following transfer to the mammalian host through sandfly bite, the parasites thrive as non-motile amastigotes within the macrophages [1,3] Earlier studies from this laboratory and others have shown that both the promastigote and the amastigote forms of Leishmania donovani are sensitive to oxidative and nitrosative stress and use specialized defense systems unique to themselves to survive when exposed to such conditions [4–7]. In the absence of an effective vaccine, limited chemotherapeutic drugs and growing drug resistance, it has become important to increase the understanding of the physiology of the Leishmania parasite to explore for new drug targets. Although several aspects of Leishmania cellular defense are known, the role of CYP450-like proteins in the survival of this parasite remains to be explored. CYP450s are hemoproteins catalysing a variety of chemical reactions including biotransformation of drugs, bioconversion of xenobiotics, chemical carcinogen metabolism and the synthesis of physiologically important compounds such as sterols and fatty acids [8]. A clear indication of CYP450 involvement in Leishmania PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25273