Effects of Clostridium difficile Toxin A on the proteome of colonocytes studied by differential 2D electrophoresis.
Effects of Clostridium difficile Toxin A on the proteome of colonocytes studied by differential 2D electrophoresis.
复制标题
DOI：
10.1016/j.jprot.2011.08.012
复制
发表时间：
2011-12
影响因子：
3.3
通讯作者：
J. Zeiser;Jennifer Klodmann;H. Braun;R. Gerhard;I. Just;A. Pich
中科院分区：
文献类型：
--
作者：
J. Zeiser;Jennifer Klodmann;H. Braun;R. Gerhard;I. Just;A. Pich
艰难梭菌是一种形成孢子的厌氧病原体，通常与严重腹泻或危及生命的假膜性结肠炎有关。其主要毒力因子为单链多结构域毒素A（TcdA）和B（TcdB）。它们的葡糖基转移酶结构域选择性地使Rho蛋白失活，导致细胞骨架的重组。为了专门研究TcdA的葡萄糖基转移酶依赖性分子效应，用重组野生型TcdA和毒素的葡萄糖基转移酶缺陷变体TcdAgd处理人结肠细胞（Caco-2）24小时。通过2-D DIGE和LCMS/MS方法结合详细的蛋白质组图谱研究结肠细胞蛋白质模式的变化。gdTcdA未诱导蛋白质模式的任何可检测的显著变化。将TcdA处理的细胞与对照组进行比较，显示出7个较高强度的斑点和2个较低强度的斑点（p<0.05）。三种蛋白质参与细胞骨架的组装（β-肌动蛋白、埃兹蛋白和DPYL 2），四种蛋白质参与代谢和/或氧化应激反应（泛素、DHE 3、MCCB、FABPL），两种蛋白质参与调节过程（FUBP 1、AL 1A 1）。这些发现与已知的TcdA效应（如细胞骨架重组）密切相关，并强调Rho蛋白糖基化对TcdA致病作用的重要性。
Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain, multi-domain toxin A (TcdA) and B (TcdB). Their glucosyltransferase domain selectively inactivates Rho proteins leading to a reorganization of the cytoskeleton. To study exclusively glucosyltransferase-dependent molecular effects of TcdA, human colonic cells (Caco-2) were treated with recombinant wild type TcdA and the glucosyltransferase deficient variant of the toxin, TcdAgdfor 24h. Changes in the protein pattern of the colonic cells were investigated by 2-D DIGE and LCMS/MS methodology combined with detailed proteome mapping. gdTcdA did not induce any detectable significant changes in the protein pattern. Comparing TcdA-treated cells with a control group revealed seven spots of higher and two of lower intensity (p<0.05). Three proteins are involved in the assembly of the cytoskeleton (β-actin, ezrin, and DPYL2) and four are involved in metabolism and/or oxidative stress response (ubiquitin, DHE3, MCCB, FABPL) and two in regulatory processes (FUBP1, AL1A1). These findings correlate well to known effects of TcdA like the reorganization of the cytoskeleton and stress the importance of Rho protein glucosylation for the pathogenic effects of TcdA.