Time-resolved cellular effects induced by TcdA from Clostridium difficile
Time-resolved cellular effects induced by TcdA from Clostridium difficile
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DOI：
10.1002/rcm.6882
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发表时间：
2014-05-30
影响因子：
2
通讯作者：
Pich, Andreas
中科院分区：
文献类型：
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作者：
Jochim, Nelli;Gerhard, Ralf;Pich, Andreas
依据：厌氧菌艰难梭菌是一种常见的病原体，可引起结肠感染，导致腹泻或假膜性结肠炎。其主要毒力因子是毒素A（TcdA）和毒素B（TcdB），其通过葡糖基化特异性地破坏小GTP酶，导致细胞骨架重组并最终导致细胞死亡。本研究采用同位素编码蛋白标记（ICPL）方法对重组野生型TcdA处理后结肠癌细胞Caco-2的蛋白质组进行了定量分析（rTcdA-wt）或葡糖基转移酶缺陷型突变体TcdA（rTcdA-mut）。方法采用液相色谱/电喷雾电离质谱（LC/ESI-MS）对粗细胞裂解液或细胞亚组分中的蛋白质进行鉴定。分析毒素处理的两个时间点（5 h，24 h），并且在每种情况下鉴定出约4000种蛋白质。在用rTcdA-wt处理5 h后，150种蛋白质具有显著改变的丰度; rTcdA-mut在该时间点引起50种蛋白质的调节。用rTcdA-wt处理24 h后，观察到61种蛋白质丰度的变化，但如果用rTcdA-mut处理细胞，则在24 h后未检测到蛋白质丰度的变化。TcdA影响参与信号事件、细胞骨架和细胞间接触组织、翻译和代谢过程的几种蛋白质。ICPL依赖的定量验证了标记的无针对性MS技术的基础上多反应监测（MRM）和三重四极杆massspectrometry.CONCLUSIONSLC/MS为基础的蛋白质组分析和ICPL方法揭示了全面和可重复的蛋白质组数据，并提供了新的见解梭菌葡萄糖基化毒素（CGT）的细胞效应。版权所有（c）2014约翰威利父子有限公司
RATIONALEThe anaerobe Clostridium difficile is a common pathogen that causes infection of the colon leading to diarrhea or pseudomembranous colitis. Its major virulence factors are toxin A (TcdA) and toxin B (TcdB), which specifically inactivate small GTPases by glucosylation leading to reorganization of the cytoskeleton and finally to cell death. In the present work a quantitative proteome analysis using the isotope-coded protein label (ICPL) approach was conducted to investigate proteome changes in the colon cell line Caco-2 after treatment with recombinant wild-type TcdA (rTcdA-wt) or a glucosyltransferase-deficient mutant TcdA (rTcdA-mut).METHODSProteins from crude cell lysates or cellular subfractions were identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Two time points (5 h, 24 h) of toxin treatment were analyzed and about 4000 proteins were identified in each case.RESULTSAfter 5 h treatment with rTcdA-wt, 150 proteins had a significantly altered abundance; rTcdA-mut caused regulation of 50 proteins at this time point. After 24 h treatment with rTcdA-wt changes in abundance of 61 proteins were observed, but no changes in protein abundance were detected after 24 h if cells were treated with rTcdA-mut. TcdA affected several proteins involved in signaling events, cytoskeleton and cell-cell contact organization, translation, and metabolic processes. The ICPL-dependent quantification was verified by label-free targeted MS techniques based on multiple reaction monitoring (MRM) and triple quadrupole mass spectrometry.CONCLUSIONSLC/MS-based proteome analyses and the ICPL approach revealed comprehensive and reproducible proteome date and provided new insights into the cellular effects of clostridial glucosylating toxins (CGT). Copyright (c) 2014 John Wiley & Sons, Ltd.