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.
McClain
M S
MS
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2605, USA.
Schraw
W
W
Ricci
V
V
Boquet
P
P
Cover
T L
TL
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