Substrate specificity of clostridial glucosylating toxins and their function on colonocytes analyzed by proteomics techniques.
Substrate specificity of clostridial glucosylating toxins and their function on colonocytes analyzed by proteomics techniques.
复制标题
DOI：
10.1021/pr300973q
复制
发表时间：
2013-03
影响因子：
4.4
通讯作者：
J. Zeiser;R. Gerhard;I. Just;A. Pich
中科院分区：
文献类型：
--
作者：
J. Zeiser;R. Gerhard;I. Just;A. Pich
艰难梭菌是医院肠道感染的主要原因。主要毒力因子是毒素A （TcdA）和毒素B (TcdB)，它们属于梭菌糖基化毒素（CGT）群，能灭活小的gtpase。与TcdA或葡萄糖基转移酶缺陷突变体TcdA （gdTcdA）孵育24小时后，使用高分辨率LC-MS/MS和SILAC技术分析结肠细胞蛋白质组（Caco-2）的定量变化。对超过5100种蛋白质的丰度变化进行了量化。发现了近800种毒素反应蛋白，这些蛋白参与细胞周期、细胞结构、粘附以及代谢过程。一些定位于线粒体或参与脂质代谢的蛋白质在TcdA处理后始终具有较高的丰度。所有蛋白质丰度的变化都取决于TcdA的糖基转移酶活性。采用LC-MS/MS检测TcdA已知靶点RhoA、RhoC、RhoG的糖基化程度。此外，Rap1（A/B）、Rap2（A/B/C）几乎完全糖基化，Ral（A/B）和（H/K/N）Ras部分糖基化。将TcdA的糖基化模式与其他CGT如TcdB、艰难梭菌菌株VPI 1470的变体TcdB （TcdBF）和梭氏梭菌致死毒素（TcsL）的糖基化模式进行比较。
Clostridium difficile is the major cause of intestinal infections in hospitals. The major virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong to the group of clostridial glucosylating toxins (CGT) that inactivate small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic cells (Caco-2) were analyzed using high-resolution LC-MS/MS and the SILAC technique. The changes in abundance of more than 5100 proteins were quantified. Nearly 800 toxin-responsive proteins were identified that were involved in cell cycle, cell structure, and adhesion as well as metabolic processes. Several proteins localized to mitochondria or involved in lipid metabolism were consistently of higher abundance after TcdA treatment. All changes of protein abundance depended on the glucosyltransferase activity of TcdA. Glucosylation of the known targets of TcdA such as RhoA, RhoC, RhoG was detected by LC-MS/MS. In addition, an almost complete glucosylation of Rap1(A/B), Rap2(A/B/C) and a partial glucosylation of Ral(A/B) and (H/K/N)Ras were detected. The glucosylation pattern of TcdA was compared to that of other CGT like TcdB, the variant TcdB from C. difficile strain VPI 1470 (TcdBF), and lethal toxin from C. sordellii (TcsL).