Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells.
Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH ('activation') results in structural changes in the protein and a marked enhancement of its cell-vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]-VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]-VacA to cells. Binding of acid-activated [125I]-VacA to cells at 4 degrees C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non-specifically or to an abundant, low-affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS-PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time-dependent process at 37 degrees C but not at 4 degrees C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]-VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2-deoxy-D-glucose. These results indicate that acid or alkaline pH-induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2605, USA.
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McClain
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Schraw
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Ricci
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Boquet
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D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism*; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015533:Transcriptional Activation; D014617:Vacuoles / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; C074689:VacA protein, Helicobacter pylori