Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells
Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells
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DOI：
10.1128/iai.64.10.4197-4203.1996
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发表时间：
1996-10-01
影响因子：
3.1
通讯作者：
Cover, TL
中科院分区：
文献类型：
--
作者：
Garner, JA;Cover, TL
许多幽门螺杆菌菌株产生细胞毒素（VacA），诱导上皮细胞空泡化。在这项研究中，HeLa或AGS（人胃腺癌）细胞的细胞毒素的结合和内化的特征在于通过间接荧光显微镜，细胞与细胞毒素在4 ℃孵育显示出均匀的荧光质膜信号。将细胞毒素与兔抗血清预孵育，以类似于90-kDa H。pylori VacA或来自H.幽门螺杆菌感染者抑制其与细胞的结合并阻断其诱导细胞质空泡化的能力。重组VacA片段（类似于34和类似于58 kDa）对应于两个类似于90 kDa VacA的蛋白酶解产物，分别与HeLa细胞的质膜结合。pylori 34-kDa片段的抗血清对细胞毒活性无影响。幽门螺杆菌细胞毒素定位于细胞内的核周位置，但不定位于细胞毒素诱导的空泡内。当细胞与先前结合的细胞毒素与抗细胞毒素血清在4 ℃孵育，然后转移到37 ℃时，空泡化被完全抑制。结合的细胞毒素在37 ° C下与细胞孵育60至120分钟后变得无法接近抗血清的中和作用。这些数据表明了一种模型，其中（i）VacA主要通过其58-kDa片段中的氨基酸序列与细胞结合，（ii）VacA内化在温度依赖性过程中缓慢发生，以及（iii）VacA与细胞内靶相互作用。
Many Helicobacter pylori strains produce a cytotoxin (VacA) that induces vacuolation in epithelial cells. In this study, binding and internalization of the cytotoxin by HeLa or AGS (human gastric adenocarcinoma) cells were characterized by indirect fluorescence microscopy, Cells incubated with the cytotoxin at 4 degrees C displayed a uniform fluorescent plasma membrane signal. Preincubation of the cytotoxin with either rabbit antiserum to similar to 90-kDa H. pylori VacA or sera from H. pylori-infected persons inhibited its binding to cells and blocked its capacity to induce cytoplasmic vacuolation. Recombinant VacA fragments (similar to 34 and similar to 58 kDa), corresponding to two proteolytic cleavage products of similar to 90-kDa VacA, each bound to the plasma membrane of HeLa cells, Antiserum reactive with the similar to 58-kDa VacA fragment inhibited the binding of native H. pylori cytotoxin to cells and inhibited cytotoxin activity, whereas antiserum to the similar to 34-kDa fragment had no effect, When incubated with cells at 37 degrees C for greater than or equal to 3 h, the H. pylori cytotoxin localized intracellularly in a perinuclear location but did not localize within cytotoxin-induced vacuoles. When cells with previously bound cytotoxin were incubated with anticytotoxin serum at 4 degrees C and then shifted to 37 degrees C, vacuolation was completely inhibited. Bound cytotoxin became inaccessible to the neutralizing effects of antiserum after 60 to 120 min of incubation with cells at 37 degrees C, These data suggest a model in which (i) VacA binds to cells primarily via amino acid sequences in its 58-kDa fragment, (ii) VacA internalization occurs slowly in a temperature-dependent process, and (iii) VacA interacts with an intracellular target.