Structural Analysis of the Helicobacter pylori Pore-Forming Toxin, VacA

Abstract

Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the stomachs of >50% of the human population, with a prevalence as high as 90% in developing nations. H. pylori infection causes gastritis and can lead to the development of peptic ulcer disease and gastric adenocarcinoma in a subset of infected individuals. Gastric cancer is a leading cause of cancer-related deaths worldwide and the World Health Organization has classified H. pylori as a type 1 carcinogen. H. pylori has evolved an arsenal of virulence factors that enhance infection, including the secreted pore forming toxin vacuolating cytotoxin A (VacA). VacA has important roles in H. pylori colonization of the human stomach and the pathogenesis of H. pylori-related gastroduodenal diseases. VacA causes multiple cellular effects including vacuolation, membrane permeabilization, mitochondrial dysfunction, cell death, and autophagy. These cellular effects are attributed to VacA oligomerizing and forming a transmembrane anion channel in various cell membranes. The mature 88-kDa VacA toxin contains two regions (p33 and p55); the N-terminal p33 region contains hydrophobic amino acids required for channel formation and sections within both p33 and p55 mediate VacA oligomerization and binding to host cells. While oligomerization and membrane insertion are essential for VacA pore forming activity, the mechanism of how VacA oligomerizes and forms a pore in cell membranes has not been fully characterized. To understand how VacA oligomerizes and associates with membrane, we determined structures of a soluble and membrane-bound VacA monomers and oligomers using single-particle cryo-electron microscopy. We mapped the regions of p33 and p55 involved in soluble VacA hexamer assembly, modeled how flexible interactions between protomers could support heptamer formation, and analyzed p33 and p55 residues that contribute to oligomerization using disulfide mutants. Additionally, we utilized negative stain and cryo-electron microscopy to structurally analyze VacA in a liposome bound and detergent-solubilized state resulting in a pre-pore structure of VacA associated with membrane. Together, this work provides structural insights into the process of VacA oligomerization and pore formation and identifies regions of VacA that undergo conformational changes upon membrane association. Since the molecular mechanisms by which VacA elicits its variety of cellular responses are not fully elucidated, these structural studies can be used to understand VacA function within the context of cells. Structural analysis of membrane proteins like VacA can be challenging, especially when imaged in presence of detergent. In this work, we identified and characterized the helical MPER-epitope tag as a strategy to complex membrane proteins with existing, easy to produce MPER-targeting antibody fragments for improved structural analysis with X-ray crystallography and single-particle electron microscopy. Altogether, this dissertation provides 1) important structural insights into how VacA oligomerizes and begins to form a pore in membrane and 2) a tool for future structural analysis of membrane proteins in general.