High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis.
Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.
Ricci
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INSERM U452, Faculté de Médecine, 28 Avenue de Valombrose, 06107 Nice Cedex 2, France.
Galmiche
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Doye
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A
Necchi
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Solcia
E
E
Boquet
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P
D008841:Actin Cytoskeleton / Q000187:drug effects; D000199:Actins / Q000187:drug effects; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D016466:CHO Cells / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology; D002460:Cell Line / Q000187:drug effects; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007457:Iodine Radioisotopes; D009761:Nystatin / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D010738:Type C Phospholipases / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002966:Clathrin; D007457:Iodine Radioisotopes; D010716:Phosphatidylinositols; D011506:Proteins; C074689:VacA protein, Helicobacter pylori; D009761:Nystatin; D015638:Cytochalasin D; D010738:Type C Phospholipases