ACID-INDUCED DISSOCIATION OF VACA, THE HELICOBACTER-PYLORI VACUOLATING CYTOTOXIN, REVEALS ITS PATTERN OF ASSEMBLY

In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form s imilar to 30-nm hexagonal ''flowers,'' each composed of an similar to 15-nm central ring surrounded by six similar to 6-nm globular ''petals .'' Upon exposure to acidic pH, these oligomeric flowers dissociate in to collections of up to 12 teardrop-shaped subunits, each measuring si milar to 6X14 nm Correspondingly, glycerol density gradient centrifuga tion shows that at neutral pH VacA sediments at similar to 22S, wherea s at acidic pH it dissociates and sediments at similar to 5S. Immunobl ot and EM analysis of the 5-S material demonstrates that it represents similar to 90-kD monomers with 6X14-nm ''teardrop'' morphology. These data indicate that the intact VacA oligomer consists of 12 similar to 90-kD subunits assembled into two interlocked six-membered arrays, ov erlap of which gives rise to the flower-like appearance. Support for t his interpretation comes from EM identification of small numbers of re latively ''flat'' oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adso rb to mica in two different orientations, corresponding to hexameric s urfaces that are either exposed or sandwiched inside the dodecamer, re spectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monom ers and the flat forms were thought to be proteolysed flowers. Since a cidification has been shown to potentiate the cytotoxic effects of Vac A, the present results suggest that physical disassembly of the VacA o ligomer is an important feature of its activation.