Effects of temperature, time, and toxin concentration on lesion formation by the Escherichia coli hemolysin

We performed osmotic protection experiments to test the hypothesis that the Escherichia coli hemolysin forms a discrete-size pore in erythrocyte membranes. The effects of toxin concentration, assay time, temperature, and protectant concentrations were examined. The results we present here raise doubts about the existing model of pore formation by hemolysin. We demonstrate that osmotic protection by various sugars of different sizes is a function of hemolysin concentration and assay time. The data indicate that under various conditions, lesion sizes with a diameter ranging from < 0.6 to > 1.2 nm can be inferred. Quantification of hemolysin permitted the estimation of the number of HlyA structural protein molecules required per erythrocyte for lysis in the presence of each protectant. It appears that hemolysin induces heterogeneous erythrocyte lesions which increase in size over time. Influx experiments utilizing radioactive sugar markers indicated that time-dependent osmotic protection patterns are independent of the diffusion rates of individual protectants. We demonstrate that the rate of the putative growth in the size of hemolysin-mediated lesions is temperature dependent. The erythrocyte membrane lesions formed at 37 degrees C can be stabilized in size when shifted to 4 degrees C. On the basis of these data, new models for the nature of the hemolysin-mediated erythrocyte membrane lesions are presented.