metabolism and bioconversion of xenobiotics. Although the parasite genome database annotations identify 3 distinct CYP450s in Leishmania donovani, no data is available on the possible functions of these proteins in this medically important parasite. Both from the point of view of identifying drug targets and understanding the physiology of the Leishmania parasite, CYP450s need to be investigated. In this study, we report the involvement of one of the CYP450s, the CYP5122A1 in the survival and function of the Leishmania donovani parasite. The sequence of the cloned CYP450 from L. donovani obtained using primers designed from L. major showed the presence of signature motifs of CYP450 proteins. Designated CYP5122A1 by the CYP450 Nomenclature Committee, the sequence of the protein showed close identity within the subgroup of Leishmania but shared ,50% identity with the Trypanosoma sequences. For functional studies, the free-swimming L. donovani promastigotes were selected as an ideal model system to analyse the function of CYP5122A1 because promastigotes differentiate into infective metacyclics that eventually infect mammalian macrophages. No significant alterations in the CYP5122A1 levels during in vitro life cycle suggested constitutive levels to be sufficient for growth and metacyclogenesis. However, the higher expression of the protein in stationary phase metacyclics as compared to the log phase ones implied that accumulation of this protein was coincident with increase in infective abilities of the parasites. Interestingly, the intracellular amastigotes also expressed a higher quantity of the protein than promastigotes. The requirement of CYP5122A1 for normal growth of the parasite was substantiated by impaired differentiation and growth of the HKO parasites where one allele of CYP5122A1 was replaced by antibiotic resistance resulting in expression of half the amount of the protein as is present in the WTs. When HKO parasites were transfected to episomally express CYP5122A1 to compensate the reduced expression in them, improved growth as compared to the HKOs further validated the requirement of CYP5122A1 for development of the parasite. The sharp decline in ergosterol levels in the HKO parasites coupled with their inability to tolerate oxidative stress indicated that the lowered ergosterol could be a reason for lesser parasite growth and lack of resistance to drug-induced stress. While the improved growth rate upon supplementation of ergosterol could be looked upon as an indication of a positive role of the sterol, the HKO parasites episomally expressing CYP5122A1 showing increased ergosterol content and improved growth rate further confirmed the importance of ergosterol. In another kinetoplastid parasite, the Trypanosoma spp. alterations in ergosterol levels through enzymatic inhibition induce reduction in cell growth [29–30]. Although Leishmania parasites can survive with lower sterol levels, normal supplement of sterol is required for optimal parasite function [17] which is reinforced by this study. Since sterol biosynthesis inhibitors are projected as attractive drug targets [16,31], lower ergosterol in the HKOs suggest a link of CYP5122A1 to sterol biosynthesis which makes a strong case for CYP5122A1 to be investigated further for evaluation as a possible drug target. Efficient ability to bind to the macrophages and invade the cells determines the extent of infection and finally, disease pathogenesis. Reduced infective ability of the HKO parasite when compared to WT parasite could be due to several reasons. Firstly, reduced infectivity could be related to lower number of infective forms present in the HKO parasite inoculum, secondly, defects in host- parasite membrane interaction or thirdly, a rapid parasite clearance by host generated ROS. The lower recovery of metacyclics from the HKO cultures as compared to the WT cultures indicated that formation of fewer metacyclics could be a reason for lower infectivity. Since metacyclics from HKOs were CYP5122A1, a Novel Cytochrome P450 of Leishmania less efficient for infection as compared to the WT metacyclics, the quality of their membrane could also be a reason for impaired ability to infect. Therefore, it is likely that a combination of factors like lesser number of infective forms in a given inoculum and defective membrane fusion events were responsible for lower infection rate by the HKO parasites. Altered lipid composition of membranes is known to affect membrane binding events in general like in yeast where ergosterols play an important role in plasma membrane fusion [32–33]. HKO parasites expressing low ergosterol were cleared faster by the host possibly because of compromised resistance to oxidative stress as sterols are proposed to be crafted by natural selection as an early defense mechanism to ROS [15]. The notion that parasites with low ergosterol are vulnerable to oxidative stress is further supported by increased susceptibility of these parasites to H2O2 induced stress in vitro. Since the extent of sterol oxidation is defined by the sterol content itself, the low ergosterol cells would be prone to increased ROS induced damage [34]. As low CYP5122A1 levels in the HKO parasites increased oxidative stress susceptibility, it was of interest to investigate how the WT parasites expressing normal CYP5122A1 levels handled drug exposure. The WT parasites resisted lower doses of miltefosine more efficiently because they could up-regulate CYP5122A1 in response to the drug. Altered lipid content by the action of miltefosine [35–36] combined with increased miltefosine-generated ROS increased the susceptibility of the HKO parasites to the drug. It was obvious that ROS played a major role in miltefosine induced cell death because presence of an antioxidant during miltefosine exposure protected the cells. In contrast, PAT treatment could not induce CYP5122A1 increase and caused higher death. Although the difference in response could be due to the dissimilar mode of action, these observations lend credence to the suggestion that CYP5122A1 levels are critical for combating drug effects. Arguably, drugs using cellular sterols to exert their action should show impaired effects in the HKO cells with lower ergosterol. Consistent with this view, HKO parasites were less susceptible to amphotericin B, an anti-leishmanial drug that requires association with membrane ergosterol for action [9,18]. This provides an important possibility that CYP5122A1 deficiency could be linked to resistance to amphotericin B as changes in sterol profile is known to accord amphotericin B resistance [9,18]. The mitochondria primarily sequesters Ca2+ to stimulate and control the rate of oxidative phosphorylation to induce mitochon- drial permeability transition, regulate cytosolic Ca2+ pulses and drive apoptotic cell death [22]. The partially functional mito- chondria, with the lower DYm, low ATP production, low Ca2+ and higher ROS generation in HKO parasites possibly reflected consequences of lower ergosterol content as well because ergosterol is found in the membranes of intracellular organelles including Leishmania mitochondria [37]. The close similarity in CYP5122A1 sequences between the old and the new world Leishmania spp. with the conserved heme binding domain suggest commonality of function with its orthologues in the Leishmania and Trypanosoma species. Therefore, how can this data be integrated in a model of Leishmania survival? The information obtained clearly establishes that interference with CYP5122A1 levels have deleterious consequences for the parasite in terms of compromised organellar function, increased suscepti- bility to drugs and impairment of the ability to infect, firmly establishing CYP5122A1 as an essential protein for Leishmania survival. Therefore, it would be of interest to check the status of this protein in Leishmania field isolates from endemic regions to relate if alterations of this protein is linked to drug resistance. The PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e25273