A Helicobacter pylori VacA subdomain required for intracellular toxin activity and assembly of functional oligomeric complexes

From the Department of Microbiology and Immunology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2605; and the Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 37212; and Department of Microbiology and Institute for Genomic Biology, University of Illinois, Urbana, IL 61801

^* To whom correspondence should be addressed. Email: timothy.L.cover{at}vanderbilt.edu.

	Abstract

Helicobacter pylori VacA is a secreted pore-forming toxin that is comprised of two domains, designated p33 and p55. The p55 domain has an important role in binding of VacA to the cell surface. About 111 residues at the amino-terminus of p55 (residues 312-422) are essential for the intracellular activity of VacA, which suggests that this region may constitute a subdomain with an activity distinct from cell binding. To investigate properties of this subdomain, a small deletion mutation (targeting aspartic acid 346 and glycine 347) was introduced into the H. pylori chromosomal vacA gene. Similar to wild-type VacA, the VacA 346-347 mutant protein was proteolytically processed, secreted, and bound to eukaryotic cells. However, VacA 346-347 did not cause cell vacuolation or membrane depolarization, and it was impaired in the ability to assemble into large water-soluble oligomeric structures. Interestingly, VacA 346-347 was able to physically interact with wild-type VacA to form mixed oligomeric complexes, and VacA 346-347 inhibited wild-type vacuolating activity in a dominant-negative manner. These data indicate that the assembly of functional oligomeric VacA complexes is dependent on specific sequences, including amino acids 346 and 347, within the p55 amino-terminal subdomain.