Icile-associated diarrhea (CDAD), a nosocomial infection with increasi…

Icile-associated diarrhea (CDAD), a nosocomial infection with increasing morbidity and mortality due to the emergence of hypervirulent strains [2,3]. Treatment with broad-spectrum antibiotics contributes to colonization of the colon with toxin producing C. difficile. The CDAD is characterized by a loss of mucosal barrier function, secretory diarrhea and colonic inflammation [4]. TcdA and TcdB are homologous single chain toxins and are composed of an N-terminally located glucosyltransferase domain and a large delivery domain. The* Correspondence: pich.andreas@mh-hannover.de Hannover Medical School, Institute of Toxicology, Carl-Neuberg-Str. 1, D30625 Hannover, Germanylatter comprises a receptor binding domain, a transmembrane domain and a cysteine protease domain [4]. The glucosyltranferase domain includes an aspartate-any amino acid-aspartate motif (D-X-D) and a conserved tryptophan that participates in the coordination of a manganese ions and the sugar donor UDP-glucose, which are essential for enzymatic glucosyltransferase activity [1]. The mutation of the D-X-D motif to N-X-N decreases glucosyltransferase activity by factor of 6,900 compared to that of wild type recombinant TcdA, so that the mutant toxin is in fact catalytically inactive PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25751659 [5]. The toxins monoglucosylate the Rho GTPases Rho, Rac, and Cdc42 and are therefore assigned as clostridial glucosylating toxins [6]. Rho proteins regulate cell morphology, gene transcription, and cell proliferation [7]. The inactivation of Rho, Rac and Cdc42 causes actin depolymerization?2011 Jochim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Jochim et al. Proteome Science 2011, 9:48 http://www.proteomesci.com/content/9/1/Page 2 ofresulting in cell rounding (cytopathic effect) and eventually leads to cell death (cytotoxic effect). Several studies reported glucosyltransferase-independent effects of TcdA on colonic cells resulting in activation of mitogen-activated protein kinases, generation of reactive oxygen species and stimulation of protein kinases PKC a and b [8-10]. The apoptotic effects have been assumed to be triggered independently of the glucosyltransferase activity. However, the studies from Gerhard et al. show a dependence on active TcdA leading to glucosylation of Rho GTPases for induction of apoptosis [11]. Thus, it is still unclear, how TcdA renders cells apoptotic. To further provide insights into these effects we investigated the changes in protein expression of epithelial colorectal adenocarcinoma cells (Caco-2) which are targets of Clostridium difficile toxins. The investigated cell line Lenalidomide is much more susceptible to TcdA than to TcdB particular if the toxin is added from the apical side as obvious in cell culture plates [12]. To compare different cellular response to wild type TcdA (rTcdA wt) and enzyme deficient mutant TcdA (mutant rTcdA) the cytosolic fractions from Caco-2 cells treated with rTcdA wt or mutant rTcdA were analyzed applying isotope-coded protein labeling (ICPLTM). ICPL is a useful and efficient approach for quantitative proteomics based on isotope tagging at free lysine residues and the N-terminus of intact proteins [13,14]. The complexity of cellular extracts was reduced by means of one-dimensional SDS-P.