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 structur al and functional alterations in eukaryotic cells. Exposure of VacA to eith er acidic or alkaline pH ('activation') results in structural changes in th e protein and a marked enhancement of its cell-vacuolating activity. Howeve r, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalizatio n of [I-125]-VacA by HeLa cells. We detected no difference in the binding o f untreated and activated [I-125]-VacA to cells. Binding of acid-activated [I-125]-VacA to cells at 4 degrees C was not saturable, and was only partia lly inhibited by excess unlabelled toxin. These results suggest that VacA b inds either non-specifically or to an abundant, low-affinity receptor on He La cells. To study internalization of VacA, we used a protease protection a ssay. 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 d etected only if VacA was acid or alkaline activated before being added to c ells. The internalization of activated [I-125]-VacA was not substantially i nhibited by the presence of excess unlabelled toxin, but was blocked if cel ls were depleted of cellular ATP by the addition of sodium azide and 2-deox y-d-glucose. These results indicate that acid or alkaline pH-induced struct ural changes in VacA are required for VacA entry into cells, and that inter nalization of the intact 87 kDa toxin is required for VacA cytotoxicity.