Functional analysis of Helicobacter pylori VacA toxin

Vanderbilt University, Nashville TN

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Abstract

DESCRIPTION (provided by applicant): Helicobacter pylori infection is a risk factor for duodenal ulcer disease and gastric cancer. One H. pylori virulence factor associated with disease is the vacuolating cytotoxin (VacA). VacA exerts a variety of effects on epithelial cells in vitro, including the formation of intracellular vacuoles and anion-selective pores in the plasma membrane. VacA domains involved in assembly of the toxin into oligomeric structures or membrane channels have not been analyzed yet. Also, domains of VacA essential for toxin binding to cells have not yet been analyzed in any detail. The aims of this proposal are: (i) identification of domain(s) required for VacA assembly into oligomeric structures. To accomplish this aim, the yeast-two hybrid method is being used. Preliminary data suggest that the yeast-two hybrid system is suitable for the screening of oligomerization domains within VacA. (ii) mapping the cell binding domain(s) required for VacA binding to target cells. To achieve this aim, a library of VacA carboxyl-terminal deletions has been generated. The mutant recombinant toxins are being expressed in E. coli and tested for their cytotoxic activity as well as their ability to bind to cells. (iii) understanding the mechanism of the dominant-negative phenotype observed with the VacA delta6-27 deletion mutant. To fulfill this aim I am using the VacA recombinant system to determine the minimum amino acid sequence required for the delta6-27-VacA toxin to exhibit a dominant-negative phenotype.