However, mass spectrometry had not been performed to unequivocally determine the mass of the native or recombinant Ehrlichia TRPs and the exact nature of the posttranslational modifications and the glycan attachment sites

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nd TcdB are not essential for the toxins biological function, albeit determining the potency of the toxin by their interactions with cell surface structures. Furthermore, we monitored huge variations in toxin potency towards different cell types as well as between the applied toxin forms. It should be noted that, contrary to other analyzed cell lines, CHO-C6 cells show identical susceptibility towards CROP-deleted and full length TcdA whereas the 3544-24-9 CROP-truncated mutant of TcdB possesses less potency towards 23370967 these cells compared to the full length toxin. This is important, because CHO cells are of hamster origin, and the Syrian hamster model is widely used for C. difficile infection models. Thus, if CHO cells are representative for hamster cells including enterocytes and colonocytes, studies on the relevance of toxins only have model character and their extrapolation to human pathogenicity is limited. Different cells/species may notedly differ in their sensitivity to TcdA and TcdB. In addition, fragments or isoforms of toxins devoid of only the C-terminal repeats are also pathogenic. The latter finding might be of epidemiologic relevance with respect to the increasing prevalence of TcdA2/TcdB+ C. difficile strains. These strains are designated as TcdA-negative though, regarding serogroup F-strains, mere lacking 1800 base pairs within the repetitive regions. The resulting truncated TcdA lacking short parts of the CROP domain is cytopathic and most likely accounts for the pathogenesis observed in the variant C. difficile strains. Author Contributions Conceived and designed the experiments: AO SG FH HT RG. Performed the experiments: AO SG FH HT RG. Analyzed the data: AO SG HT RG. Contributed reagents/materials/analysis tools: RG IJ HT. Wrote the paper: AO RG. Critical discussion: IJ. 13 March 2011 | Volume 6 | Issue 3 | e17623 CROP-Mediated Endocytosis of TcdA 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. and B; evidence that Ca2+ plays a role in toxin A cell surface association. J Mol Biol 346: 1197206. Pothoulakis C, Gilbert RJ, Cladaras C, Castagliuolo I, Semenza G, et al. Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A. J Clin Invest 98: 64149. Na X, Kim H, Moyer 20534789 MP, Pothoulakis C, LaMont JT gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A. Infect Immun 76: 2862871. Just I, Gerhard R Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 152: 237. Pruitt RN, Chagot B, Cover M, Chazin WJ, Spiller B, et al. Structurefunction analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A. J Biol Chem 284: 219341940. Amimoto K, Noro T, Oishi E, Shimizu M A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C. Microbiology 153: 1198206. Burger S, Tatge H, Hofmann F, Just I, Gerhard R Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system. Biochem Biophys Res Commun 307: 58488. Jainchill JL, Aaronson SA, Todaro GJ Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol 4: 54953. Hidalgo IJ, Raub TJ, Borchardt RT Characterization of the human colon carcinoma cell line as a model system for intestinal epithelial permeability. Gastroenterology 96: 73649. Huet C, Sahuquillo-Merino C, Coudrier E, Louvard D Absorptive and mucus-secreting subclones isolated from a multipote