Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism
Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism
复制标题
DOI：
10.1016/j.jprot.2011.11.018
复制
发表时间：
2012-02-16
影响因子：
3.3
通讯作者：
Burchmore, Richard
中科院分区：
文献类型：
--
作者：
Daneshvar, Hamid;Wyllie, Susan;Burchmore, Richard
我们先前已经描述了婴儿利什曼原虫（H-线）的减毒线，通过在庆大霉素的存在下在体外培养前鞭毛体选择。为了阐明这种衰减的分子基础，我们进行了比较蛋白质组学分析，使用多重二维（2D）差异凝胶电泳。18个蛋白质，表现出显着的和可重复的表达变化进行了鉴定。其中许多是利什曼原虫的硫醇-氧化还原控制系统的组成部分，这一观察结果，通过Western blot验证，促使我们研究衰减线对氧化应激的敏感性。发现减毒品系对过氧化氢明显更敏感，这一变化可能解释了毒力丧失的原因。在锥虫硫酮依赖性过氧化物酶活性的直接测定中，H系中的过氧化氢代谢显著低于野生型。此外，锥虫硫酮还原酶活性显着较低的H-线，表明庆大霉素的选择可能会导致多效性影响利什曼原虫的硫醇代谢。一个假定的RNA结合蛋白是非常强烈的上调减毒线，这表明庆大霉素在利什曼原虫的可能目标。(C)2011 Elsevier B. V.保留所有权利。
We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania. (C) 2011 Elsevier B.V. All rights reserved.