Sequential Binding of Staphylococcal y-Hemolysin to Human Erythrocytes and Complex Formation of the Hemolysin on the Cell Surface†

Staphylococcal gamma-hemolysin consists of two protein components, F (or H gamma I) and H gamma II. To elucidate the mode of action of gamma-hemolysin, we studied the binding order of F and H gamma II to human erythrocytes and the cell-bound state of the two components. The binding of F to human erythrocytes preceded the binding of H gamma II to the cells, and thereafter hemolysis occurred. Western immunoblot analysis of the cell-bound gamma-hemolysin indicated that F and H gamma II components form high-molecular-mass (150-250 kDa) complexes on the erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins. Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K. Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of H gamma II to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes.