Cell Death and Differentiation Research Laboratory, Centre for Molecular Medicine National Institute of Immunology, New Delhi, India.
PLoS One. 2011;6(9):e25273. doi: 10.1371/journal.pone.0025273. Epub 2011 Sep 23.
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 Ca(2+) 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.
细胞色素 P450s（CYP450s）是催化多种生化反应的血红素蛋白，这些反应对于外来物质的代谢和固醇等生理重要化合物的合成至关重要。因此，它们对于入侵病原体的生存具有重要的功能。其中一种机会性病原体利什曼原虫（Leishmania donovani）导致全世界的内脏利什曼病，由于疾病负担重大，这是一个重要的公共卫生问题。寄生虫基因组数据库 Gene DB 在利什曼原虫中注释了 3 种 CYP450，然而，细胞色素 P450 酶在利什曼原虫中的功能作用仍不清楚。
方法/主要发现：从利什曼原虫中克隆的一种 CYP450 样基因被鉴定为一种新型 CYP450，即 CYP5122A1。通过与细胞器特异性标记物的共定位，显示 CYP5122A1 分布定位于前鞭毛体 ER、线粒体和糖基体。通过同源重组将 CYP5122A1 的一个等位基因用新霉素或潮霉素基因替换，在前鞭毛体中诱导 CYP5122A1 表达的显著减少。这些寄生虫的生长受到损害，线粒体 Ca（2+）和膜电位降低，导致 ATP 生成减少。此外，与野生型相比，这些寄生虫在体外和体内的感染性降低，这通过体外与巨噬细胞孵育利什曼原虫前鞭毛体以及通过将寄生虫给药到仓鼠中来评估。HKO 对米替福新和锑等药物更敏感，但对两性霉素 B 的敏感性降低。去除 CYP5122A1 的两个等位基因不允许寄生虫存活。与野生型寄生虫相比，突变寄生虫中的麦角固醇水平低 3.5 倍，通过气相色谱/质谱法估计。通过使用 pXG-GFP+2 载体通过质体表达蛋白来补充 CYP5122A1，部分挽救了 CYP5122A1 的表达，并将麦角固醇水平恢复了 1.8 倍。表型逆转包括恢复生长模式和降低药物敏感性。
结论/意义：总之，这项研究确立了 CYP5122A1 作为与利什曼原虫中的细胞生长、感染和麦角固醇生物合成等过程相关的重要分子。
