Structural Analysis of the Helicobacter pylori Toxin VacA

Abstract

CELL AND DEVELOPMENTAL BIOLOGY Structural Analysis of the Helicobacter pylori toxin VacA Tasia Marie Pyburn Dissertation under the direction of Associate Professor Melanie Ohi

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. One of the most important H. pylori virulence determinants is a secreted pore-forming toxin known as vacuolating cytotoxin A (VacA). Secreted as an 88 kDa protein, VacA is composed of an N-terminal p33 domain and a C-terminal p55 domain which assemble into multiple types of water-soluble oligomers including hexamers, heptamer, dodecamers, and tetradecamers. We have determined three-dimensional (3D) structures of VacA s1/i1/m1 oligomeric conformations at ~15 Å resolution as well as three mutant forms of VacA. At this resolution, differences between the mutants and VacA s1/i1/m1 could not be discerned. Therefore, cryo-EM has been performed on VacA s1/i1/m1 and a structure has been determined of a VacA dodecamer to the highest resolution to date, ~10Å resolution. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. Our goal is to understand how VacA transitions from a soluble protein to a membrane inserted protein and how it organizes on membrane. Using a combination of in vitro liposome binding, biochemical assays, and single particle electron microscopy (EM), we show membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle EM reveals structural differences within the central pore-forming region of the oligomers indicating that membrane interactions induce a structural change within the p33 domain. Analyses of VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.

Approved _______________________________________________ Date __________________ Melanie D. Ohi, Ph.D.