Abstract
Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryoelectron microscopy, and in the current study, we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effects of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative-stain electron microscopy and single-particle two-dimensional class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization, and cell vacuolation. Internalization of a nonoligomerizing mutant form of VacA by host cells was detected without a requirement for acid activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.