Department of Developmental Medicine, Osaka Medical Center for Maternal and Child Health, Research Institute, Osaka, Japan.
FEMS Microbiol Lett. 2011 May;318(1):10-7. doi: 10.1111/j.1574-6968.2011.02233.x. Epub 2011 Mar 2.
The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.
小分子寡聚淀粉样中间体能形成非特异性离子通道，对宿主细胞膜有毒性。耐热直接溶血素（TDH）和 TDH 相关溶血素（TRH）是副溶血性弧菌的主要毒力因子。我们之前已经报道了 TDH 四聚体的晶体结构，其中包含中央通道。我们还确定了 TDH 对热处理的矛盾反应（即 Arrhenius 效应）的分子机制，这种反应是可逆的淀粉样变性特性。在本报告中，我们描述了 TRH 的生物物理特性，TRH 与 TDH 具有 67%的氨基酸相似性。分子建模为 TDH 和 TRH 的整体结构提供了很好的拟合。凝胶过滤层析、超速离心和透射电子显微镜显示 TRH 在溶液中形成四聚体。这些毒素在红细胞上显示出相似的溶血活性。然而，与 TDH 相比，通过硫黄素 T 结合试验和远紫外圆二色光谱分析，TRH 的类淀粉样结构较少。这些数据表明，加热时的淀粉样变性对于红细胞的膜破坏并非必不可少，但维持四聚体结构对于 TDH 和 TRH 的溶血活性是必不可少的。
