Patel Hetal K, Willhite David C, Patel Rakhi M, Ye Dan, Williams Christopher L, Torres Eric M, Marty Kent B, MacDonald Robert A, Blanke Steven R
Department of Biology and Biochemistry, University of Houston, Texas 77204-5001, USA.
Infect Immun. 2002 Aug;70(8):4112-23. doi: 10.1128/IAI.70.8.4112-4123.2002.
The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using beta-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, beta-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.
幽门螺杆菌空泡毒素(VacA)在体外和体内均可诱导哺乳动物细胞发生退行性空泡化。在此,我们证明质膜胆固醇对于VacA诱导的哺乳动物细胞空泡化至关重要。当使用β-环糊精从质膜中选择性提取胆固醇时,多种细胞系中的空泡生物发生被完全阻断。此外,提高质膜胆固醇水平可显著增强VacA诱导的空泡化。相反,用洛伐他汀或康帕丁抑制胆固醇的从头生物合成对空泡化没有可检测到的影响。虽然已表明质膜胆固醇的耗尽会干扰网格蛋白介导的内吞作用和小窝依赖的内吞作用,但这两种内化途径均未被发现对VacA诱导的细胞空泡化至关重要。耗尽质膜胆固醇会减弱VacA进入HeLa细胞的过程。此外,β-环糊精试剂可阻断预先加载VacA或在胞质溶胶中直接表达VacA的细胞的空泡化。总体而言,我们的结果表明质膜胆固醇对于VacA的中毒机制和随后的空泡生物发生均很重要。