CYP5122A1, a Novel Cytochrome P450 of Leishmania Figure 2. Expression of CYP5122A1 in amastigotes and promastigotes of L. donovani. A: Western blot analysis of equal protein quantities from whole cell lysates prepared from L. donovani amastigotes (A) and promastigotes (P) probed with anti-CYP5122A1 antibody. B: Western blots of total cell lysates prepared from L. donovani promastigotes at different time points during an in vitro growth cycle, probed with anti-CYP5122A1 antibody. Loading was normalized with Tubulin–a (50 kDa). C: Bar graph; right hand y-axis represents quantitation of immunoblots shown in B, mean 6 SE, n = 3. Line graph, left hand y-axis represents the number of metacyclic parasites formed during the in vitro growth cycle. D: Western blot analysis of whole cell lysates prepared from L. donovani metacyclic prepared from logarithmic (L) and stationary (S) phase culture probed with anti- CYP5122A1 antibody indicating marginal up-regulation of CYP5122A1. doi:10.1371/journal.pone.0025273.g002 slower than the WT cells showing a slower growth curve (Fig. 5B and Fig. S4E). HKO parasites in general were less motile unlike the highly motile WTs. Constitutive ROS level was higher in the HKOs as compared to WTs (Fig. 5C) when measured on various days of cycle. Impaired mitochondrial function in the HKOs was evident from lower mitochondrial membrane potential (DYm) and lower ATP levels as compared to WTs (Fig. 5D and E). Mitochondrial Ca2+, a known inducer of the generation of DYm as well as the utilization of the ATP [22] was also lower in the HKO parasites (Fig. 5F). As evident from the above data, reduced expression of CYP5122A1 in the promastigotes compromised growth and mitochondrial function. Knockdown of CYP5122A1 compromises the parasites ability to infect both in vitro and in vivo Next, we sought to compare the infective abilities of the WT and the HKO parasites to assess if lesser CYP5122A1 expression would impair the parasites’ ability to infect. Parasites containing the pBSK+ Neo cassette as replacement of one allele of CYP5122A1 showed reduced infecting abilities as compared to WT cells in terms of attachment at 2, 4 and engulfment at 6 h (Fig. 6A–C). Similarly, parasites containing the pBSK+Hyg cassette also showed a reduced ability to infect (Fig. S4G; % macrophages infected, control, 7466; hygromycin HKO, 2263, n = 3, *P#0.05). The engulfed HKO parasites were cleared within 10 h of infection as suggested by the reduction in the number of macrophages containing internal parasites (Fig. 6B). This could be due to clearance by the macrophage generated ROS where HKO parasites would be more susceptible to such stress. This is further supported by the increased death of HKO promastigotes in vitro as compared to WTs when exposed to H2O2 (Fig. S5A). To assess the number of metacyclics in the inoculum of the HKO parasites to see if low number of metacyclics reduced infective abilities, peanut agglutinin based separation was used to isolate the infective forms. Lower numbers of metacyclics were recovered from the HKO cultures as compared to WT cultures (Fig. 7A) suggesting lesser differentiation if one allele of CYP5122A1 was removed. When the total number of metacyclics in the HKO inoculum was raised by increasing multiplicity of infection, there was a moderate increase in the number of macrophages infected but the infection was comparatively lower than infection achieved with the WT parasites (Fig. S5B). To investigate if the difference in infection rate was only due to the lesser number of metacyclics in the HKO cultures, macrophages were infected with equal numbers of metacyclics alone, isolated from both the WT and the HKO cultures. The infection pattern observed was similar to that obtained with total cellular cultures (Fig. 7B–D). This clearly showed that the HKO parasites cultures produced fewer metacyclics that were comparatively less efficient to infect as compared to metacyclics isolated from WT cultures. Subsequently, to confirm the in vitro infection data in vivo, hamsters were used as a model system. Eight weeks after parasite administration to adult hamsters, significant splenomegaly was observed in animals infected with WT parasites as compared to the spleens in hamsters infected with HKO parasites as reflected in the comparative spleen weight (Fig. 7E). In consonance with the above observations, microscopic counts of splenic smears from HKO infected hamsters showed lesser number of macrophages containing parasites whereas splenic smears from WT parasite infected hamsters showed a large number of macrophages loaded with parasites (Fig. 7F). This data suggested that in the absence of full constitutive expression of CYP5122A1, the parasites demon- strated a compromised ability to infect both in vitro and in vivo. Knockdown of CYP5122A1 alters the parasites ability to withstand drug pressure In the absence of vaccines, the treatment of visceral leishmaniasis has centred around several drugs like sodium stibogluconate (pentavalent antimony), amphotericin B and more recently, miltefosine [23]. The HKO parasites showed increased susceptibility to potassium antimony tartrate (PAT) the active form of pentavalent antimony and miltefosine (Fig. 8A–B, Fig. S4F) suggesting a role of CYP5122A1 in drug response. Therefore, based on the aforementioned results, WT parasites were checked for cellular level of CYP5122A1 after drug treatment. PAT was unable to induce any change in CYP5122A1 levels (Fig. 8C, (i) and (ii) whereas miltefosine was able to induce an up- PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25273