VacA subdomain required for intracellular toxin activity and 1 assembly of functional oligomeric complexes 2 3

17 Helicobacter pylori VacA is a secreted pore-forming toxin that is comprised of two 18 domains, designated p33 and p55. The p55 domain has an important role in binding of VacA to 19 the cell surface. About 111 residues at the amino-terminus of p55 (residues 312-422) are 20 essential for the intracellular activity of VacA, which suggests that this region may constitute a 21 subdomain with an activity distinct from cell binding. To investigate properties of this 22 subdomain, a small deletion mutation (targeting aspartic acid 346 and glycine 347) was 23 introduced into the H. pylori chromosomal vacA gene. Similar to wild-type VacA, the VacA 24 ∆346-347 mutant protein was proteolytically processed, secreted, and bound to eukaryotic cells. 25 However, VacA ∆346-347 did not cause cell vacuolation or membrane depolarization, and it was 26 impaired in the ability to assemble into large water-soluble oligomeric structures. Interestingly, 27 VacA ∆346-347 was able to physically interact with wild-type VacA to form mixed oligomeric 28 complexes, and VacA ∆346-347 inhibited wild-type vacuolating activity in a dominant-negative 29 manner. These data indicate that the assembly of functional oligomeric VacA complexes is 30 dependent on specific sequences, including amino acids 346 and 347, within the p55 amino-31