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J Biol Chem, Vol. 274, Issue 53, 37736-37742, December 31, 1999
From the § Departments of Medicine and
Most Helicobacter pylori
strains secrete a toxin (VacA) that causes structural and functional
alterations in epithelial cells and is thought to play an important
role in the pathogenesis of H. pylori-associated
gastroduodenal diseases. The amino acid sequence, ultrastructural
morphology, and cellular effects of VacA are unrelated to those of any
other known bacterial protein toxin, and the VacA mechanism of action
remains poorly understood. To analyze the functional role of a unique
strongly hydrophobic region near the VacA amino terminus, we
constructed an H. pylori strain that produced a mutant VacA
protein (VacA-(
A Dominant Negative Mutant of Helicobacter pylori
Vacuolating Toxin (VacA) Inhibits VacA-induced Cell Vacuolation*
,
,,§**
Microbiology and Immunology, Vanderbilt University School
of Medicine and ** Veterans Affairs Medical Center,
Nashville, Tennessee 37232, the Department of Biology and
Biochemistry, University of Houston, Houston, Texas 77204, and the
¶ Department of Molecular Physiology and Biological Physics
and Biophysics Program, University of Virginia School of Medicine,
Charlottesville, Virginia 22908
6-27)) in which this hydrophobic segment was
deleted. VacA-(6-27) was secreted by H. pylori,
oligomerized properly, and formed two-dimensional lipid-bound crystals
with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of
wild-type VacA and VacA-(6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce
cell vacuolation. Moreover, when an equimolar mixture of purified
VacA-(6-27) and purified wild-type VacA were added simultaneously
to HeLa cells, the mutant toxin exhibited a dominant negative effect,
completely inhibiting the vacuolating activity of wild-type VacA. A
dominant negative effect also was observed when HeLa cells were
co-transfected with plasmids encoding wild-type and mutant toxins. We
propose a model in which the dominant negative effects of
VacA-(6-27) result from protein-protein interactions between the
mutant and wild-type VacA proteins, thereby resulting in the formation
of mixed oligomers with defective functional activity.
*
This work was supported in part by National Institutes of
Health Grants AI39657, DK53623 (to T. C.), RR07720, HL48807 (to Z. S.), and R37 HL37127 (to G. S.), the American Heart Association (to Z. S. and S. B.), an Oak Ridge Junior Faculty Enhancement award
(to S. B.), and the Department of Veterans Affairs (to T. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Infectious Diseases, A3310 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-2035; Fax: 615-3436160; E-mail: COVERTL@CTRVAX.VANDERBILT.EDU.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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