Analysis of nanobodies reactive with Helicobacter pylori VacA toxin
Helicobacter pylori secretes a pore-forming toxin called VacA. VacA enhances the ability of H. pylori to colonize the gastric mucosa and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease. The secreted VacA toxin is an 88 kDa protein that is comprised of two domains, p33 and p55. The p55 domain is primarily a beta-helical structure, and plays an essential role in facilitating VacA binding to gastric epithelial cells. The amino terminal portion of the p33 domain is required for formation of membrane channels and cytotoxic activity. A high resolution structure of the p33 domain has not yet been determined. Membrane channel formation and cytotoxic effects of VacA are dependent on oligomerization of the protein. To facilitate studies of VacA structure and function, a panel of VacA-reactive single-domain antibodies (nanobodies) has been generated. In this study, I will analyze the reactivity of these nanobodies with multiple forms of VacA. Thus far, I have transformed E. coli with plasmids encoding the relevant nanobodies, and I have purified the nanobodies produced by these transformants. When tested by ELISA, at least 8 unique nanobodies react with VacA oligomers. Further experiments will map the sites in VacA that are recognized by these nanobodies and will test the nanobodies for toxin-neutralizing activity. These experiments will provide further insight into the structure and function of VacA.