Helicobacter pylori remains a significant human pathogen and is linked to gastric disease, including peptic ulcers and gastric cancer. A key determinant in H. pylori pathogenesis is the presence of the VacA toxin, which induces vacuolation in gastric epithelial cells. It is hypothesised that VacA forms intracellular oligomeric anion-selective channels that lead to chloride ion leakage. Our lab has identified that VacA undergoes proteolytic processing in human cells, with cleavage of a hydrophilic loop region that contributes to its activation. The molecular nature of these active VacA channels, however, remains unknown; for example, how processing of the hydrophilic loop region affects oligomerisation and channel activity.
In this study, we generated a mutant variant of VacA (DI) targeting the hydrophilic loop region, which was observed to have remarkably lower vacuolating activity compared to wildtype VacA following intoxication of AGS cells. Moreover, we used mass photometry to demonstrate that the DI mutant has a higher propensity for oligomerisation compared to wild-type VacA, suggesting that this variant has altered the equilibrium for self-association. We performed single particle analysis cryo-EM which confirmed that the DI mutant retains the overall VacA structure. Next, we analysed the intracellular processing which was noticeable different in the DI form which further supports that the hydrophilic loop is involved in the formation of the active anion channel. Indeed, we suggest that the hydrophilic loop is involved in mediating the processing of distal regions such as the C-terminal foot of VacA. Further investigation into the role of the hydrophilic loop will provide crucial insights into the mechanisms of VacA proteolytic activation. This knowledge may be beneficial for developing new therapeutics to reduce H. pylori associated diseases, such as gastric cancer.