Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin

Abstract

The binding of Staphylococcus aureus alpha-toxin to rabbit and human erythrocytes was studied by hemolytic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Hemolytic assays showed that toxin binding to 10% cell suspensions at neutral pH was very ineffective in the concentration range 3 X 10(-8) to 3 X 10(-7) M (1 to 10 micrograms/ml), and less than 5% of added toxin became cell bound. However, binding was augmented as toxin levels were raised, abruptly increasing to 50 to 60% at 2 X 10(-6) to 3 X 10(-6) M (60 to 100 micrograms/ml). When rabbit erythrocytes were lysed with 1 to 5 micrograms of toxin per ml, both monomeric and hexameric forms of the toxin could be detected on the membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. In contrast, human erythrocytes treated with 1 to 6 micrograms of toxin per ml did not lyse, and membrane-bound toxin was not detectable. When toxin concentrations were raised to 30 to 100 micrograms/ml, human erythrocytes also lysed and toxin hexamers became membrane bound in comparable amounts as on rabbit cell membranes. Lowering the pH led to a marked increase in susceptibility of human, but not rabbit erythrocytes towards alpha-toxin. When human cells were lysed at pH 5.0 with 5 micrograms of toxin per ml, membrane-bound hexameric toxin became detectable. The demonstrated correlation between the presence of hexameric, cell-bound toxin and hemolytic activity supports the channel concept of toxin-mediated cytolysis. The results also show that toxin binding does not exhibit overall characteristics of a simple receptor-ligand interaction.