Tam Patricia J, Lingwood Clifford A
Departments of Laboratory Medicine and Pathobiology, University of Toronto, Canada.
Research Institute, Program in Molecular Structure and Function, The Hospital for Sick Children, Ontario M5G 1X8, Canada.
Microbiology (Reading). 2007 Aug;153(Pt 8):2700-2710. doi: 10.1099/mic.0.2007/006858-0.
In sensitive cells, verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [125I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA1, was demonstrated for the first time by monitoring [125I]VTA1 release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA1 and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA1 detection. Brefeldin A and 16 degrees C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA1 cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA1 values indicates that about one molecule of translocated VTA1 per cell is sufficient to inhibit protein synthesis and kill a cell.
在敏感细胞中，志贺毒素1（VT1）利用一种依赖于球三糖神经酰胺受体的逆行转运途径，从细胞表面转运至高尔基体/内质网（ER）。VT1 A亚基（VTA）是一种RNA聚糖酶。尽管VTA从内质网转运至胞质溶胶被认为是蛋白质合成抑制的途径，但缺乏基于细胞的证据。构建了一种双荧光标记的VT1全毒素，以同时监测VTA和VT1 B亚基（VTB）的细胞内转运。通过共聚焦显微镜观察，VTA/VTB亚基在整个逆行转运途径中保持结合状态，未出现胞质染色。然而，在经[125I]VT1处理的细胞中，通过监测经链球菌溶血素O（SLO）使质膜通透后[125I]VTA1的释放，首次证明了活化形式的VTA即VTA1存在选择性的胞质转运（4%）。蛋白酶体抑制剂乳胞素可增加胞质中的VTA1，并增强VT1的细胞毒性。VT1到达内质网与胞质中VTA1的检测同时发生。布雷菲德菌素A和16℃这两种抑制VT1向高尔基体/内质网逆行转运的条件，可阻止VTA1的胞质转运；然而，这些处理并未完全阻止VT1诱导的蛋白质合成抑制。因此，VTA1有效的胞质转运需要转运至高尔基体/内质网，但当向高尔基体/内质网的转运被阻止时，可能存在其他次要的蛋白质合成抑制逃逸途径。蛋白质合成抑制具有时间和剂量依赖性，不一定是后续细胞病理学的有效指标。仅在VT1暴露超过3小时后出现的蛋白质合成抑制与最终的细胞毒性相关。对转运至胞质的VTA1值进行推断表明，每个细胞中约一个转运的VTA1分子就足以抑制蛋白质合成并杀死一个细胞。
