A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B
A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B
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DOI：
10.1371/journal.pntd.0007260
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发表时间：
2019-04-01
影响因子：
3.8
通讯作者：
Madhubala, Rentala
中科院分区：
文献类型：
--
作者：
Bansal, Ruby;Sen, Shib Sankar;Madhubala, Rentala
背景杜氏利什曼原虫是一种原生动物寄生虫，是内脏利什曼病的主要病原体。通过甲氧戊酸途径产生的甾醇，在不同的生物界显示出不同的组成。22-不饱和甾醇在真菌中以麦角甾醇的形式存在，在植物中以豆甾醇的形式存在，在侧链的C-22位置上含有双键。在本研究中，我们报道了杜氏利什曼原虫中一个类似植物的细胞色素P450亚家族细胞色素P450亚家族CYP710C1的鉴定和功能鉴定，该基因是利什曼原虫C-22脱饱和酶。测定了重组细胞色素P710C1作为C-22去饱和酶的酶功能。为了进一步研究CYP710C1在利什曼原虫中的生理作用，我们培育并鉴定了一株高表达菌株和一株基因缺失突变体。在野生型、高表达的前鞭毛体和杂合子突变体中，我们估计了C-22去饱和酶的活性和豆甾醇的水平。结论我们首次报道了在利什曼原虫中存在一个编码类似于甾醇C-22去饱和酶的植物的CYP710C1基因，该基因导致豆甾醇的生物合成。重组细胞色素P710C1具有将谷甾醇转化为豆甾醇的C-22去饱和酶活性。无菌无鞭毛体的CYP710C1mRNA、蛋白和豆甾醇的表达水平均高于前无鞭毛体。对高表达杜氏乳杆菌和杂合突变寄生虫的CYP710C1进行的甾醇分析表明，该基因负责豆甾醇的产生。最重要的是，我们证明了这些过度表达的前鞭毛体对两性霉素B具有抗药性，两性霉素B是治疗利什曼病的首选药物。我们报道利什曼原虫甾醇生物合成途径具有嵌合组织，兼有植物和真菌途径的特征。
Background Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of 22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase.Methodology In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants.Conclusion We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting -sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways.