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首页> 外文期刊>Cell biology international. >Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes.
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Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes.

机译：大肠杆菌肠溶血素的生产，表征及其对红细胞膜结构的影响。

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摘要

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.
机译：溶血素是由病原菌产生的细胞破坏性蛋白毒素，通常释放到细胞外培养基中。大肠杆菌肠溶血素是在生长的对数期产生的一种细胞内毒素，在对数后期达到最大的细胞内蓄积。在本研究中，我们已采用电子显微镜和SDS-PAGE评估肠溶血溶血素对不同物种红细胞膜的影响。暴露于血红蛋白溶血素后立即开始红细胞损伤，化学上可检测到细胞膜通透性的变化，表面损伤的形成迅速增大。该过程导致细胞完全破坏。用肠溶血溶血素处理马红细胞膜后，用电子显微镜观察到直径为10nm的环形结构。发现环结构成簇且不规则地分布在膜表面上。在将毒素与马红细胞重影孵育并溶解去污剂后，通过蔗糖密度梯度以膜结合形式从细胞质中分离出肠溶血溶素。 SDS-PAGE和脱氧胆酸盐溶解的目标膜的银染显示毒素的异质形式。通过使用SDS-PAGE和凝胶过滤，毒素的分子量估计为35 kDa。就物种特异性而言，马红细胞对肠溶血素的敏感性最高，其次是人和豚鼠的红细胞。溶血敏感性与不同动物物种的红细胞膜的毒素结合能力有关。溶血度不受温度在4摄氏度至37摄氏度范围内的影响，在pH 9.0时最佳。与成孔的溶细胞素相反，在5至30 mM范围内增加浓度的葡聚糖4和葡聚糖8的存在下，肠溶血素的溶血活性不断增强。膜结合肠溶血溶血素的胰蛋白酶敏感性表明细胞表面是该毒素最可能的靶位。另外，蛋白酶和磷酸酶抑制剂不能抑制裂解的事实表明肠溶血素通过去污剂样机制改变和破坏细胞膜。

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