Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin -Susceptible -Resistant Leishmania donovani
Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin -Susceptible -Resistant Leishmania donovani
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DOI：
10.1371/journal.pone.0026660
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发表时间：
2011-10-27
期刊：
影响因子：
3.7
通讯作者：
Madhubala, Rentala
中科院分区：
文献类型：
--
作者：
Chawla, Bhavna;Jhingran, Anupam;Madhubala, Rentala
多诺瓦利什曼原虫是一种引起内脏利什曼病（VL）的原生动物寄生虫，可导致严重的死亡率和发病率。抗锑药物耐药性的增加对VL的化疗提出了巨大的挑战。帕罗霉素是一种氨基糖苷类抗生素，是目前用于皮肤和内脏利什曼病化疗的药物之一。为了在分子水平上了解这种抗生素的作用方式，我们研究了野生型AG83菌株和多诺瓦尼L. paromomycin resistant （PRr）菌株之间的全球蛋白质组差异。采用细胞培养氨基酸稳定同位素标记（SILAC）和定量质谱法对野生型AG83和paromomycin resistant （PRr）菌株进行鉴定，在>= 95%置信区间共鉴定出226种蛋白质。数据分析显示，PRr菌株中29个蛋白表达上调，21个蛋白表达下调。野生型和paromomycin耐药菌株的比较蛋白质组学分析显示，耐药菌株的核糖体蛋白上调，表明其参与翻译。在PRr菌株中也观察到糖酵解酶和应激蛋白水平升高。最重要的是，我们观察到PRr菌株中可能在细胞内存活和囊泡运输中起作用的蛋白质上调。此外，电镜超微结构分析表明，与野生型菌株相比，PRr菌株的囊泡数量增加。用药物亲和力下拉法和质谱法鉴定了多诺瓦氏L.野生型菌株中与帕罗霉素特异性共价结合的蛋白。这些结果为多诺瓦利什曼原虫对帕罗霉素耐药的作用方式和潜在机制提供了第一个全面的见解。
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at >= 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.