title: Substrate specificity of clostridial glucosylating toxins and their function on colonocytes analyzed by proteomics techniques.
abstract: 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).
authors: Hannover Medical School, Institute of Toxicology , Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors: Johannes
authors: J
authors: Zeiser
authors: Ralf
authors: R
authors: Gerhard
authors: Ingo
authors: I
authors: Just
authors: Andreas
authors: A
authors: Pich
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism*; D016360:Clostridioides difficile / Q000378:metabolism / Q000472:pathogenicity; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006031:Glycosylation; D054884:Host-Pathogen Interactions; D006801:Humans; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D011506:Proteins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; C075397:RHOG protein, human; C116756:RAP2A protein, human; C066690:RHOC protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein