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 (10to 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 (2to 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.

Review article: tolevamer, a novel toxin‐binding polymer: overview of preclinical pharmacology and physicochemical properties

“…These toxins are known to alter cell metabolism and inhibit protein synthesis. 27,28 In animal models, the individual toxins are capable of causing severe colitis, similar to that seen with CDAD. 11,13 Tolevamer apparently binds these toxins in the colon, thereby blocking their ability to interact effectively with the intestinal epithelium.…”

Purification and Some Properties of Cytotoxin Produced by Clostridium difficile

“…The other toxin is cytotoxin or toxin B which shows cytotoxicity to the cultured cells. Many investigators (2,9,11,16,17,23,24,26) focused their study on enterotoxin to determine characteristics which might relate to causing diarrhea and other clinical features. They established the method of purification of enterotoxin and characterized its properties.…”

“…It has been reported that this organism produced at least two kinds of toxin (2,4,17,21,(24)(25)(26) ; one is a toxin named toxin A or enterotoxin which induces an intestinal fluid accumulation in animal experiments (2,16,17,26), and the other is toxin B or cytotoxin which is cytotoxic on tissue cultured cells (2,16,20,24,26). Several investigators including us (2,9,11,23,24) have established the purification method of enterotoxin, and the importance of this toxin in the pathogenesis of C. difficile enteritis has been documented (18,26). Data on the cytotoxin reported up to now, however, are inconsistent.…”

Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells

“…which are implicated in the pathogenesis of the colitis (10)(11)(12)(13)16). Enterotoxin A (EA) causes accumulation of fluid in ligated rabbit ileal loops (11,13), whereas cytotoxin B appears to have primarily a cytotoxic effect on tissue culture cells (12,17,20,28). The toxins have a complex effect on cultured cells and intestinal tissue, including changes in ionic balance and morphology of the cells (11,17,20).…”