CYP5122A1, a Novel Cytochrome P450 of Leishmania times lesser ergosterol than the WTs as evident from GC-MS based analysis of organic solvent extracts of the cells (Fig. 9A). Although it is not known if the Leishmania parasite can incorporate ergosterol in vitro, a related kinetoplastid parasite, Trypanosoma brucei takes up sterol precursors in the host [27]. Ergosterol supplement- ed in vitro during Leishmania culture showed an uptake of ergosterol, and a higher HKO parasite growth rate in contrast to those cultured without ergosterol supplementation (Fig. 9B). Presence of a higher level of ergosterol in CYP5122A1 complemented HKO parasites indicated CYP5122A1 involvement in maintaining ergosterol levels, although the extent of recovery was not equivalent to the WTs (Fig. 9A). The alteration of sterol levels could have important implication in drug response because ergosterol is required for action of some drugs. For example, amphotericin B, a polyene antifungal drug also used for the treatment of leishmaniasis [1] associates with ergosterol for its action [28], arguably therefore, cytotoxic effect of amphotericin B would be lesser in the HKO parasites. There was a distinct lowering of amphotericin B cytotoxicity in CYP5122A1 HKOs in comparison to the WTs (Fig. 9C) as evident from the percentage viabilities. Therefore, this data clearly indicated that deficiency in CYP5122A1 resulted in lowering of ergosterol levels consequently making them less susceptible to amphotericin B. Discussion Cytochrome P450s are an important group of proteins involved in the synthesis of physiologically active compounds, drug Figure 9. Synthesis of ergosterol in HKO and WT parasites. A. Chromatogram of ergosterol profile from sterol extracts of WT, HKO and CMPL strains subjected to GC-MS analysis. Insets. Table showing the approximate ergosterol content as calculated using a standard curve. TLC analysis of ergosterol from WT (Lane 1), HKO (Lane 2) and complemented parasites (Lane 3). Rf value of ergosterol: 0.588. Standard ergosterol dilutions (Lane 4: 0.05 mg, Lane 5: 0.1 mg, Lane 6: 0.5 mg, Lane 7: 1 mg). B. Comparative growth pattern of WT and HKO parasites in vitro over a period of 6 days in culture, in the presence and absence of ergosterol. Mean 6 SE, n = 3. C. Bar graph shows percentage viability of WT, HKO and CMPL parasites in response to treatment with amphotericin B for 6 h. Mean 6 SE, n = 3. doi:10.1371/journal.pone.0025273.g009 PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25273