Zeiser Johannes, Gerhard Ralf, Just Ingo, Pich Andreas
Hannover Medical School, Institute of Toxicology , Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
J Proteome Res. 2013 Apr 5;12(4):1604-18. doi: 10.1021/pr300973q. Epub 2013 Mar 4.
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).
艰难梭菌是医院肠道感染的主要病因。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）组，可使小GTP酶失活。在用TcdA或糖基转移酶缺陷型突变体TcdA（gdTcdA）孵育24小时后，使用高分辨率液相色谱-串联质谱（LC-MS/MS）和稳定同位素标记氨基酸细胞培养技术（SILAC）分析结肠细胞（Caco-2）蛋白质组的定量变化。对5100多种蛋白质丰度的变化进行了定量。鉴定出近800种毒素反应蛋白，它们参与细胞周期、细胞结构、黏附以及代谢过程。TcdA处理后，几种定位于线粒体或参与脂质代谢的蛋白质丰度持续较高。蛋白质丰度的所有变化均取决于TcdA的糖基转移酶活性。通过LC-MS/MS检测到TcdA已知靶标的糖基化，如RhoA、RhoC、RhoG。此外，还检测到Rap1（A/B）、Rap2（A/B/C）几乎完全糖基化，以及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）和索氏梭菌的致死毒素（TcsL））的糖基化模式进行了比较。
