title: Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile.
abstract: Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000633:toxicity*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003013:Clostridium / Q000472:pathogenicity*; D003603:Cytotoxins / Q000633:toxicity*; D004734:Energy Metabolism / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D011188:Potassium / Q000378:metabolism; D014176:Protein Biosynthesis
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; C031815:toxB protein, Clostridium difficile; D011188:Potassium