The binary toxin produced by Clostridium botulinum enters cells by receptor-mediated endocytosis to exert its...

The binary toxin produced by Clostridium botulinum produced dose-dependent changes in cell morphology, characterized by loss of extensions and rounding. The effects were obtained when both polypeptide chains of the toxin were added to cells at 37 degrees C; the effects were inhibited by low temperature (4 degrees C) and by sodium azide (15 mM). Attachment of the heavy polypeptide chain to cells before the light polypeptide chain was necessary to produce cell poisoning. When cells were maintained at 4 degrees C, the receptor-bound heavy chain remained at the cell surface and accessible to the light chain. When cells were maintained at 37 degrees C, the receptor-bound heavy chain was internalized and no longer accessible to light chain. Drugs that antagonize receptor-mediated endocytosis, such as ammonium chloride and methylamine hydrochloride, inhibited internalization of the heavy chain-light chain complex. These drugs did not antagonize receptor binding by the heavy chain or did they inhibit mono(ADP-ribosyl)ation in broken cells by the light chain. However, they did diminish the amount of mono(ADP-ribosyl)ation produced by holotoxin in intact cells. The data indicate that receptor-mediated endocytosis is an essential step in the ability of the toxin to poison cells.