Sommi P, Ricci V, Fiocca R, Necchi V, Romano M, Telford J L, Solcia E, Ventura U
Institute of Human Physiology, University of Pavia and Istituto Ricovero e Cura a Carrattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy.
Am J Physiol. 1998 Oct;275(4):G681-8. doi: 10.1152/ajpgi.1998.275.4.G681.
The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.
空泡毒素A（VacA）是幽门螺杆菌导致人胃上皮细胞损伤的最重要毒力因子之一。利用培养的人胃上皮细胞和产VacA的幽门螺杆菌菌株的肉汤培养滤液，我们研究了：1）VacA向细胞的递送；2）内化毒素的定位和归宿；3）毒素在细胞内的持续性。所采用的研究技术包括中性红染料摄取、超微结构免疫细胞化学、定量免疫荧光和免疫印迹法。我们发现VacA：1）以游离和膜结合形式（即细菌外膜形成的囊泡）递送至细胞；2）以游离和膜结合形式定位于内体-溶酶体区室；3）在细胞内持续存在至少72小时，且不丧失空泡形成能力，但只有加入氯化铵时这种能力才变得明显；4）一般不会降解为小于约90 kDa的片段。我们的研究结果表明，虽然大量VacA在内体-溶酶体区室中积累，但它避免了主要的溶酶体降解过程并保持了其明显的分子完整性。
