Department of Bacteriology, Nagasaki University, Nagasaki, Japan.
Glycobiology. 2010 Jun;20(6):668-78. doi: 10.1093/glycob/cwq014. Epub 2010 Jan 28.
Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.
神经节苷脂是细菌进入的靶受体，但存在于人乳中的神经节苷脂对细菌感染表现出保护作用。在这里，我们表明，用浓度为 100μg/ml 的神经节苷脂混合物处理可显著抑制胃上皮癌细胞 AZ-521 中幽门螺杆菌空泡形成毒素（VacA）的空泡形成活性。研究的所有神经节苷脂（GM1、GM2、GM3、GD1a、GD1b、GD3 和 GT1b）均显示出对 VacA 的良好中和能力。通过用溶酶体 GM1 偶联到琼脂糖上作为标记多糖聚合物进行下拉测定，从幽门螺杆菌培养上清液中捕获 VacA。GM1-VacA 复合物成功沉淀，表明 GM1 直接结合 VacA。荧光相关光谱法测量的溶酶体 GM1 和 VacA 的流体动力学结合具有 190nm 的 K(d) 值。VacA 也在 pH2 下与溶酶体 GM1 结合，这对应于人体胃的生理 pH 值。总之，这些结果表明幽门螺杆菌 VacA 与游离神经节苷脂的直接结合中和了 VacA 的毒素活性。这些发现为神经节苷脂在 VacA 毒性和幽门螺杆菌发病机制中的作用提供了另一种见解。
