B-164. Assembly of Helicobacter pylori VacA into Large Oligomeric Structures Requires Amino Acid Sequences within the p33 Domain

C. Gonzalez-Rivera1, M. S. McClain1, T. L. Cover2;
1Vanderbilt Univ., Nashville, TN, 2Vanderbilt Univ. and VAMC, Nashville, TN.

Background: An important virulence factor produced by Helicobacter pylori is a secreted toxin known as VacA. This 88 kDa toxin consists of two domains, designated p33 and p55. VacA monomers assemble to form large water soluble oligomers, and VacA oligomerization is required for membrane channel formation. Many VacA-induced cellular alterations arise as a result of VacA oligomerization, insertion into membranes, and membrane channel formation. A previous study (Genisset et al., 2006) reported that a VacA mutant protein containing a 9-amino-acid deletion within the p33 domain (VacA Δ 49-57) failed to assemble into oligomeric structures. In this study, we sought to investigate further which amino acids within the p33 domain are required for formation of oligomers. Methods: Four different mutations (resulting in deletion of VacA amino acid 53, amino acids 53-57, amino acids 49-53, and amino acids 51-55) were introduced into the H. pylori chromosomal vacA gene. Mutants were characterized by PCR, restriction enzyme analysis, and nucleotide sequencing to verify that the desired mutations were present. We then analyzed VacA protein expression and secretion by Western blotting, tested the effects of each mutant VacA protein on HeLa cells, and analyzed the oligomerization of mutant VacA proteins by gel filtration chromatography. Results: Each of the mutant strains expressed and secreted a VacA protein that was about 88 kDa under denaturing conditions. When tested on HeLa cells, the Δ 53 mutant protein caused vacuole formation similar to wild-type VacA, whereas the Δ 53-57, Δ 49-53, and Δ 51-55 mutant proteins failed to cause vacuole formation. Wild-type VacA and the Δ 53 mutant protein each assembled into large oligomeric structures, whereas the Δ 53-57, Δ 49-53, and Δ 51-55 mutant proteins were defective in assembly into large oligomers. Conclusion: These data help to define specific amino acids within the p33 domain that are required for VacA oligomerization and vacuolating toxin activity. We propose a model in which the assembly of VacA into large oligomeric structures depends on intermolecular interactions between p33 and p55 domains.