Moayeri M, Welch R A
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison 53706.
Infect Immun. 1994 Oct;62(10):4124-34. doi: 10.1128/iai.62.10.4124-4134.1994.
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.
我们进行了渗透保护实验，以检验大肠杆菌溶血素在红细胞膜上形成特定大小孔道的假说。研究了毒素浓度、测定时间、温度和保护剂浓度的影响。我们在此呈现的结果对现有的溶血素孔道形成模型提出了质疑。我们证明，不同大小的各种糖类的渗透保护作用是溶血素浓度和测定时间的函数。数据表明，在各种条件下，可以推断出直径范围从<0.6到>1.2纳米的损伤大小。溶血素的定量分析使得能够估计在每种保护剂存在的情况下，每个红细胞裂解所需的HlyA结构蛋白分子数量。溶血素似乎诱导了异质性的红细胞损伤，且损伤大小会随时间增加。利用放射性糖类标记物进行的内流实验表明，时间依赖性的渗透保护模式与各个保护剂的扩散速率无关。我们证明，溶血素介导的损伤大小的假定增长速率是温度依赖性的。当从37摄氏度转移到4摄氏度时，在37摄氏度形成的红细胞膜损伤大小可以稳定下来。基于这些数据，提出了关于溶血素介导的红细胞膜损伤性质的新模型。
