A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA)inhibits VacA-induced cell vacuolation

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structu ral and functional alterations in epithelial cells and is thought to play a n important role in the pathogenesis of H. pylori-associated gastroduodenal diseases, The amino acid sequence, ultrastructural morphology, and cellula r effects of VacA are unrelated to those of any other known bacterial prote in toxin, and the VacA mechanism of action remains poorly understood. To an alyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H pylori strain that produced a muta nt VacA protein (VacA-(Delta 6-27)) in which this hydrophobic segment was d eleted. VacA-(Delta 6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features t hat were indistinguishable from those of wild-type VacA However, VacA-(Delt a 6-27) formed ion-conductive channels in planar Lipid bilayers significant ly more slowly than did wild-type VacA and the mutant channels were less an ion-selective. Mixtures of wild-type VacA and VacA-(Delta 6-27) formed memb rane channels with properties intermediate between those formed by either i solated species, VacA-(Delta 6-27) did not exhibit any detectable defects i n binding or uptake by HeLa cells, but this mutant toxin failed to induce c ell. vacuolation, Moreover, when an equimolar mixture of purified VacA(Delt a 6-27) and purified wild-type VacA were added simultaneously to HeLa cells , the mutant toxin exhibited a dominant negative effect, completely inhibit ing the vacuolating activity of wild-type VacA A dominant negative effect a lso was observed when HeLa cells were cotransfected with plasmids encoding wild-type and mutant toxins. me propose a model in which the dominant negat ive effects of VacA-(Delta 6-27) result from protein-protein interactions b etween the mutant and wild-type VacA proteins, thereby resulting in the for mation of mixed oligomers with defective functional activity.