title: pH-induced conformational changes in Clostridium difficile toxin B.
abstract: Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.
authors.affiliation: Department of Botany and Microbiology, The University of Oklahoma, Norman, Oklahoma 73019, USA.
authors.forename: M
authors.initials: M
authors.lastname: Qa'Dan
authors.forename: L M
authors.initials: LM
authors.lastname: Spyres
authors.forename: J D
authors.initials: JD
authors.lastname: Ballard
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry*; D016466:CHO Cells; D016360:Clostridioides difficile / Q000737:chemistry*; D006224:Cricetinae; D003603:Cytotoxins / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012697:Serine Endopeptidases / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018942:Macrolides; D009282:Naphthalenesulfonates; C031815:toxB protein, Clostridium difficile; C040929:bafilomycin A1; D014364:Tryptophan; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D006180:Proton-Translocating ATPases