Genomewide Analysis of Mode of Action of the S-Adenosylmethionine Analogue Sinefungin in Leishmania infantum
Genomewide Analysis of Mode of Action of the S-Adenosylmethionine Analogue Sinefungin in Leishmania infantum
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DOI：
10.1128/msystems.00416-19
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发表时间：
2019-09-01
期刊：
影响因子：
6.4
通讯作者：
Ouellette, Marc
中科院分区：
文献类型：
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作者：
Bhattacharya, Arijit;Sharma, Mansi;Ouellette, Marc
为了进一步了解原生动物寄生虫利什曼原虫的一碳代谢，我们进行了基因组筛选，以研究寄生虫如何响应 Sinefungin (SNF) 选择。 SNF 是 S-腺苷甲硫氨酸 (AdoMet) 的结构类似物，AdoMet 是许多生物分子的关键甲基供体。一项筛选包括对通过逐步增加药物浓度的逐步选择产生的 SNF 抗性突变体进行测序。这些研究证明，通过基因间重组删除 AdoMet 转运蛋白 (AdoMetT1) 是 SNF 抗性的重要功能丧失标记。第二次筛选由 Cosseq 组成，这是一种基于粘粒的功能获得性全基因组功能筛选，具有增加的 SNF 浓度与下一代测序相结合。在该筛选和测序中富集的粘粒导致鉴定出 (i) AdoMet 合成酶 (METK) 作为主要 SNF 靶标，(ii) mRNA [(鸟嘌呤-N7)-甲基转移酶 (CMT1)1，(iii) 亮氨酸羧基甲基转移酶 (LCMT)，(iv) 两个胰蛋白酶基因，以及 (v) 两种蛋白磷酸酶 调控基因。进一步的功能探索表明，LCMT 与一个磷酸酶催化亚基 (PP2AC) 相互作用，并且 PP2AC C 端亮氨酸残基的突变影响辛芬净的敏感性。这些整体筛选导致了利什曼原虫转运蛋白、生物合成基因、RNA 和蛋白质甲基转移酶以及与 AdoMet 介导功能相关的磷酸酶的鉴定。 重要性 两种主要的细胞代谢一碳供体是还原叶酸和 S-腺苷甲硫氨酸，其生物合成途径已被证明在各种化疗干预中非常有效。 细胞类型。 Sinefungin 是 S-腺苷甲硫氨酸的核苷类似物，已被证明对原生动物寄生虫利什曼原虫具有有效的活性。在这里，我们使用全基因组方法研究了对辛芬净的耐药性，以进一步了解 S-腺苷甲硫氨酸在这种寄生虫中的作用并揭示新的潜在药物靶点。这些方法可以表征利什曼原虫中与 S-腺苷甲硫氨酸功能相关的新特征，这可以进一步帮助开发针对这种病原寄生虫的类似 Sinefungin 的化合物。
To further our understanding of one-carbon metabolism in the protozoan parasite Leishmania, we conducted genomic screens to study how the parasite responded to sinefungin (SNF) selection. SNF is a structural analogue of S-adenosylmethionine (AdoMet), a key methyl group donor to a number of biomolecules. One screen consisted of sequencing SNF-resistant mutants generated by step-wise selection with gradually increasing drug concentrations. These studies demonstrated deletion of the AdoMet transporter (AdoMetT1) by intergenic recombination as a crucial loss-of-function marker for SNF resistance. The second screen consisted of Cosseq, a gain-of-function cosmid-based genomewide functional screen with increasing SNF concentration coupled to next-generation sequencing. Cosmids enriched in that screen and sequenced led to the identification of (i) the AdoMet synthetase (METK) as the major SNF target, (ii) an mRNA [(guanine-N7)-methyltransferase (CMT1)1, (iii) a leucine carboxyl methyltransferase (LCMT), (iv) two tryparedoxin genes, and (v) two protein phosphatase regulatory genes. Further functional exploration indicated that LCMT interacts with one phosphatase catalytic subunit (PP2AC) and that mutation of the C-terminal leucine residue of PP2AC affects sinefungin susceptibility. These holistic screens led to the identification of transporters, biosynthetic genes, RNA and protein methyltransferases, as well as phosphatases linked to AdoMet-mediated functions in Leishmania.IMPORTANCE The two main cellular metabolic one-carbon donors are reduced folates and S-adenosylmethionine, whose biosynthetic pathways have proven highly effective in chemotherapeutic interventions in various cell types. Sinefungin, a nucleoside analogue of S-adenosylmethionine, was shown to have potent activity against the protozoan parasite Leishmania. Here, we studied resistance to sinefungin using whole-genome approaches as a way to further our understanding of the role of S-adenosylmethionine in this parasite and to reveal novel potential drug targets. These approaches allowed the characterization of novel features related to S-adenosylmethionine function in Leishmania which could further help in the development of sinefungin-like compounds against this pathogenic parasite.