CYP5122A1, a Novel Cytochrome P450 of Leishmania Figure 7. HKOs are impaired in metacyclogenesis and their ability to infect in vivo. A. Number of WT and HKO metacyclics during in vitro life cycle represented by bar graph. Mean 6 SE, n = 3, * P#0.05. B: Bar graph showing percentages of macrophages infected with WT and HKO metacyclics at an MOI of 1:10. Mean 6 SE, n = 3, * P#0.05. C: Number of parasites present in infected macrophages when infection was carried out with equal number of WT and HKO metacyclics. Mean 6 SE, n = 3, * P#0.05. D: Photomicrographs of macrophage infected with metacyclics (arrows) isolated from WT and HKO parasite cultures. Scale, 10 mm. E: Bar graph shows spleen weights of hamsters infected for 8 weeks. Control: uninfected hamsters; WT: hamsters infected with WT parasites, HKO: hamsters infected with HKO parasites. Mean 6 SE ,n = 3, * P#0.05. F. Infected macrophage numbers represented as the number of infectious foci per field in splenic smears from hamsters infected with WT (WT) and HKO parasites (HKO). Data represents mean 6 SE, n = 5; in which 10 different fields were analysed. * P#0.05. doi:10.1371/journal.pone.0025273.g007 with CYP5122A1 complementation, the complemented parasites were comparatively more resistant to drugs as compared to the HKOs but not as efficient as WTs to resist drug induced death (Fig. S6E). This fusion protein also localized in the ER of the parasites as seen by a co-localization with ER Tracker blue white DPX stain (Fig. S6F). Changes in ergosterol occur with alterations in CYP5122A1 levels Since several CYP450s are functionally related to sterol biosynthesis [26], the HKOs carrying one allele of CYP5122A1 were checked for ergosterol content. HKO parasites expressed 3.5 PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25273