Effects of Staphylococcal Alpha-, Beta-, Delta-, and Gamma-Hemolysins on Human Diploid Fibroblasts and HeLa Cells: Evaluation of a New Quantitative Assay for Measuring Cell Damage

Abstract

Human diploid embryonic lung fibroblasts and HeLa cells were cultivated in Eagle minimaĺ essential medium supplemented with 10% calf serum. Monolayer cultures were labeled with 3 H-uridine and treated with highly purified staphylococcal alpha-, beta-, delta-, or gamma-hemolysin. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane after treatment with each hemolysin. The assay method described is simple, sensitive, and rapid. It allows quantitative estimation of changes in membrane permeability to be detected before a morphological damage is observed microscopically. Upon incubation for up to 30 min with highly purified staphylococcal hemolysins, only delta-hemolysin caused release of a significant amount of tritiated substances from fibroblasts. Such leakage occurred immediately after addition of delta-lysin and was independent of temperature. With minor exceptions, this was similar to the release of isotopes after treatment of the cells with the nonionic detergent Triton X-100. Treatment of fibroblasts with combinations of two or three of these toxins gave neither a synergistic nor an antagonistic effect. Evidence is presented which indicates that delta-hemolysin is the only important fibroblast damaging activity in crude preparations of extracellular proteins of four strains of S. aureus , whereas HeLa cells are susceptible also to purified alpha-toxin.