Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly

doi:10.1083/jcb.138.4.759

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© The Rockefeller University Press, 0021-9525/1997//759 $5.00
The Journal of Cell Biology, Volume 138, Number 4, , 1997 759-769

Article

Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly

Timothy L. Cover^*, Phyllis I. Hanson, and John E. Heuser

^* Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, and the Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232-2605; Department of Pharmacology, Yale University, New Haven, Connecticut 06510; and Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

In this study, we describe the ultrastructural changes associatedwith acid activation of Helicobacter pylori vacuolating cytotoxin(VacA). Purified VacA molecules imaged by deep-etch electronmicroscopy form $~$ 30-nm hexagonal "flowers," each composed of an $~$ 15-nm central ring surrounded by six $~$ 6-nm globular "petals."Upon exposure to acidic pH, these oligomeric flowers dissociateinto collections of up to 12 teardrop-shaped subunits, eachmeasuring $~$ 6 x 14 nm. Correspondingly, glycerol density gradientcentrifugation shows that at neutral pH VacA sediments at $~$ 22S, whereas at acidic pH it dissociates and sediments at $~$ 5 S. Immunoblot and EM analysis of the 5-S material demonstratesthat it represents $~$ 90-kD monomers with 6 x 14–nm "teardrop"morphology. These data indicate that the intact VacA oligomerconsists of 12 $~$ 90-kD subunits assembled into two interlockedsix-membered arrays, overlap of which gives rise to the flower-likeappearance. Support for this interpretation comes from EM identificationof small numbers of relatively "flat" oligomers composed ofsix teardrop-shaped subunits, interpreted to be halves of thecomplete flower. These flat forms adsorb to mica in two differentorientations, corresponding to hexameric surfaces that areeither exposed or sandwiched inside the dodecamer, respectively.This view of VacA structure differs from a previous model inwhich the flowers were interpreted to be single layers of sixmonomers and the flat forms were thought to be proteolysedflowers. Since acidification has been shown to potentiate thecytotoxic effects of VacA, the present results suggest thatphysical disassembly of the VacA oligomer is an important featureof its activation.

Address all correspondence to Timothy L. Cover, Division ofInfectious Diseases, A3310 Medical Center North, VanderbiltUniversity School of Medicine, Nashville, TN 37232. Tel.: (615)322-2035. Fax: (615) 343-6160.

This project was supported by U.S. Public Health Service grantsGM-29647 (J. Heuser), AI-39657, and R29 DK-45293 (T. Cover)by Human Frontiers Science Program grant RG355/94 (J. Heuser),by the Department of Veterans Affairs (T. Cover), and by a postdoctoralfellowship from the Helen Hay Whitney Foundation (P. Hanson).

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