RECOVERY OF HUMAN FIBROBLASTS FROM ATTACK BY THE PORE-FORMING ALPHA-TOXIN OF STAPHYLOCOCCUS-AUREUS

When applied at low concentrations (<10 mu g/ml), staphylococcal alpha -toxin generates a small channel in keratinocyte and lymphocyte membra nes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 mu g/ml) of alpha-toxin s imilarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemente d with serum and at pH 7.4, the cells are able to recover from toxin a ttack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the presence of serum in the medium and is very sensitive towards pH. Decreases of pH in th e medium to less than or equal to 7.0 as well as increases to greater than or equal to 7.8 causes the repair mechanism to fail. The fate of cell-bound toxin molecules was investigated by using a radiolabelled t racer and by immunological detection of toxin exposed at the cell surf ace. The results indicated that 50-70% of the toxin was shed from cell membranes. However, there was no clear correlation between shedding a nd recovery, and shedding was also observed in cells that died at pH 7 .8. Shedding was not decisive for repair, since cells that had recover ed from toxin attack continued to carry 30-40% of initially bound toxi n on their cell surface. Blockade of Na+/K+-ATPases with ouabain evoke d similar kinetics of K+-depletion in control cells, compared with cel ls that had just recuperated from toxin attack and that still carried 30-40% alpha-toxin on their surface. We therefore tentatively conclude d that repair of alpha-toxin lesions was due to closure of small pores , rather than from compensation of membrane leaks by upregulation of N a+/K+-ATPase activity. We speculate that repair of small membrane lesi ons may extend to other agents that produce channels of similar nature in nucleated cells. Larger pores created by E. coil hemolysin or stre ptolysin O, both of which form larger functional transmembrane lesions , could not be repaired by fibroblasts.