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 variet y of cultured cell Lines, sensitivity to VacA differing greatly, however, a mong 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 glycosylphosphatidylinosito l (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function o f 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-negat ive mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant: of dynamin II) did not affect vacuo lation induced by VacA. Nevertheless, F-actin depolymerization, known to bl ock the different types of endocytic mechanisms, strongly impaired VacA vac uolating activity. Taken together, our data suggest that the high cell sens itivity to VacA defends on the presence of one or several GPI-anchored prot ein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin -independent endocytic pathway.