May 7, 1984 - generating discrete transmembrane channels. The mem- brane-bound form of the toxin was isolated and identified as a ring-structured toxin ...
Vol. 46, No. 2
INFECTION AND IMMUNITY, Nov. 1984, P. 318-323
0019-9567/84/110318-06$02.00/0 Copyright © 1984, American Society for Microbiology
Correlation Between Toxin Binding and Hemolytic Activity in Membrane Damage by Staphylococcal oa-Toxin SUCHARIT BHAKDI,* MARION MUHLY, AND ROSWITHA FUSSLE
Institute of Medical Microbiology, University of Giessen, D-6300 Giessen, Federal Republic of Germany Received 7 May 1984/Accepted 6 July 1984
The binding of Staphylococcus aureus a-toxin to rabbit and human erythrocytes was studied by hemolytic 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 ,ug/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 ,ug/ml). When rabbit erythrocytes were lysed with 1 to 5 ,ug 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 ,ug of toxin per ml did not lyse, and membrane-bound toxin was not detectable. When toxin concentrations were raised to 30 to 100 ,ug/ 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 a-toxin. When human cells were lysed at pH 5.0 with 5 ,ug of toxin per ml, membranebound 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. assays
detergent (5). Finally, hexamers also formed upon spontanebinding of the toxin to human plasma low-density lipoprotein (6). Hence, toxin binding and hexamer formation did not appear to require the presence of specific biological receptor molecules such as a membrane (glyco)protein or glycolipid. In this respect, ot-toxin contrasted with the group of sulfhydryl-activated bacterial cytolysins, which specifically bind to cholesterol (19). Nevertheless, several salient aspects of toxin-membrane interaction are still unclear at present. Thus, it has been reported that rabbit erythrocytes do possess a small number of specific, saturable binding sites for radioiodinated toxin which are absent on human erythrocytes (10). This might explain the puzzling relative resistance of human cells toward toxin-mediated hemolysis (17). Further, rabbit erythrocytes lysed by low concentrations of toxin appeared to carry no characteristic ultrastructural rings corresponding to the hexamers. It was therefore suggested that the formation and function of the toxin oligomers might be quite unrelated to the mechanism through which membrane damage occurs (10). This would place obvious constraints on the proposed channel concept of toxin action (13). Finally, a major discrepancy appeared to exist between the early binding data for ox-toxin, which indicated that only very small quantities became cell bound (
