Senescent erythrocytes exhibit a single-hit response to staphylococcal alpha toxin

In vitro aged rabbit erythrocytes are 2- to 3-fold less sensitive than fresh erythrocytes to lysis by the alpha toxin of Staphylococcus aureus. Previously, we correlated the loss in hemolytic sensitivity as cells aged with the proteolytic degradation of Band 3, a putative binding site for staphylococcal alpha toxin. Here we separated young and old erythrocytes by density gradient centrifugation and compared their patterns of lysis by alpha toxin. Derivative plots of lytic-time curves exhibited two lytic peaks for young erythrocytes versus a single peak for the aged erythrocytes. Hit analysis showed that the first peak in the young cells was generated by the monomeric form of the toxin while the second peak involved the polymeric form. Similar analysis of the single peak in the aged erythrocytes showed that only a monomeric form of the toxin is involved in the lysis. Furthermore, analysis of alpha toxin receptor (Band 3) indicated that the receptor is gradually lost as erythrocytes age. This loss in Band 3 is proportional to the decrease in the rate of lysis by alpha toxin. Based on these comparisons we suggest that the intact Band 3 promotes polymeric assembly of the alpha toxin which becomes disrupted by the age-related changes occurring in the receptor.