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Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

Department of Microbiology and Immunology,¹ Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605,² Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212³

Received 1 November 2005/ Returned for modification 5 December 2005/ Accepted 6 January 2006

The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated 6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length 6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (6-27p33) did not exhibit detectable inhibitory activity. The 6-27p48 protein physically interacted with wild-type p88 VacA, whereas the 6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either 6-27p33 or (6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

Editor: J. T. Barbieri

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