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 - PubMed (original) (raw)

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

V Ricci et al. Mol Biol Cell. 2000 Nov.

Free PMC article

Abstract

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.

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Figures

Figure 1

Figure 1

Neutral red uptake by HEp-2 and CHO cells variously exposed to VacA. (A) Cells were incubated for either 5 h or 16 h at 37°C with VacA+ BCF or purified VacA in HBSS containing 5 mM NH4Cl (see Methods). (B) Cells were exposed to VacA+ BCF or purified VacA in HBSS for 1 h at 4°C and then, after extensive washing, incubated for either 5 h or 16 h in HBSS containing 5 mM NH4Cl (see Methods). Controls were paired monolayers not exposed to VacA. Means ± SEM of three independent experiments. * = p < 0.05 versus paired Control. ° = p < 0.05 versus the same condition at 5 h.

Figure 2

Figure 2

Effect of PI-PLC treatment on VacA-induced vacuolation of HEp-2 cells. Neutral red uptake was measured either 1) in cells previously exposed to VacA pulse (both as VacA+ BCF and as purified VacA) and then treated or not with PI-PLC (VacA preloaded), or 2) in cells treated or not with PI-PLC and then exposed or not to VacA pulse (VacA afterloaded) (see Methods). Incubation for vacuole development: 3 h at 37°C in HBSS containing 5 mM NH4Cl. Means ± SEM of three independent experiments.* = p < 0.05 versus paired monolayers not exposed to VacA.

Figure 3

Figure 3

Ultrastuctural features of HEp-2 cells treated or not with PI-PLC and then exposed to VacA. Cells untreated (A) or treated with PI-PLC (B) were incubated for 10 min with VacA+ BCF and then for 3 h with HBSS containing 5 mM NH4Cl (see Methods). Note large vacuoles filled with intense VacA immunoreactivity in A, to be compared with the small vacuoles and scarce VacA immunoreactivity in B. Aldehyde-osmium fixation, VacA immunogold, uranyl-lead counterstaining. Bars = 0.85 μm.

Figure 4

Figure 4

Effect of nystatin (Nyst) treatment on VacA-induced vacuolation of HEp-2 cells. Neutral red uptake was measured either 1) in cells previously exposed to VacA+ BCF pulse, or in control cells and then treated with Nyst dissolved in DMSO or with DMSO alone (VacA preloaded); or 2) in cells treated with Nyst dissolved in DMSO, or with DMSO alone and then exposed or not (Control) to VacA+ BCF pulse (VacA afterloaded) (see Methods). Incubation for vacuole development: 5 h at 37°C in HBSS containing 5 mM NH4Cl. Means = SEM of three independent experiments.* = p < 0.05 versus paired Control. ° = p < 0.05 versus the same condition but DMSO-treated.

Figure 5

Figure 5

VacA-induced vacuolation in HEp-2 cells overexpressing proteins specifically inhibiting the formation of clathrin-coated vesicles. HEp-2 cells grown on coverslip were transiently transfected with vectors allowing overexpression of 1) a dominant-negative mutant of the Eps15 protein, tagged with GFP (GFP-Edelta95/295) (A-D); 2) the five tandem SH3 domains of intersectin, tagged with GFP (GFP-Intersectin-SH3) (E-H); and 3) the K44A dominant-negative mutant of dynamin II, tagged with a HA epitope (HA-Dynamin II K44A) (I-L). Transfected monolayers were exposed to VacA+ BCF pulse, incubated for 16 h at 37°C in HBSS containing 3 mM NH4Cl, and then tested for TxR-Transferrin endocytosis as described in Methods. After fixations, cells were processed for immunofluorescence to detect Rab7-positive vacuoles and, where required, HA tag. (A, E, I): transfected cells (green). (B, F, J): TxR-Transferrin (red). (C, G, K): Rab7 localization (cyan). (D, H, L): merge images showing several Rab7-positive vacuoles in transfected cells where there was virtually no uptake of TxR-Transferrin. Bars = 10 μm.

Figure 6

Figure 6

Effect of cytochalasin D (CD) treatment on VacA-induced vacuolation of HEp-2 cells. Neutral red uptake was measured either 1) in cells previously exposed to VacA+ BCF pulse, or in control cells and then treated with CD dissolved in DMSO or with DMSO alone (VacA preloaded); or 2) in cells treated with CD dissolved in DMSO, or with DMSO alone and then exposed or not (Control) to VacA+ BCF pulse (VacA afterloaded) (see Methods). Incubation for vacuole development: 5 h at 37°C in HBSS containing 5 mM NH4Cl. Means = SEM of three independent experiments. * = p < 0.05 versus paired Control. ° = p < 0.05 versus the same condition but DMSO-treated.

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