Structural Characterization of the Helicobacter Pylori VacA Toxin by Single Particle Em and X-Ray Crystallography

Abstract

Helicobacter pylori is a Gram-negative bacterium that infects the human stomach and contributes to the pathogenesis of peptic ulceration, gastric adenocarcinoma and gastric lymphoma. H. pylori secretes an exotoxin called vacuolating toxin (VacA), known for its ability to induce vacuolation in the cytoplasm of mammalian cells. VacA can cause depolarization of membrane potential, alteration of mitochondrial membrane permeability, apoptosis, activation of mitogen-activated protein kinases, inhibition of T cell activation and proliferation, and autophagy. The mechanisms by which these processes occur are not yet fully understood but many of these toxic effects depend on the capacity of VacA to form anion-selective membrane channels. VacA is an 88 kDa protein that contains two distinct domains, p55 and p33. The 88 kDa monomers can assemble into large water-soluble oligomeric “flower”-shaped structures. Using single particle electron microscopy and the random conical tilt approach, we have determined three-dimensional (3D) structures of six distinct VacA oligomeric conformations at ∼15 A resolution. This analysis shows that VacA can organize into a number oligomeric conformations that include both single and double layer hexamers and heptamers. The structures, regardless of oligomeric type, contain two prominent features: extended straight “legs” with a slight kink at the distal end and a central “spoke-like” density that contains two distinct globular domains separated by a thin connecting density. We have also generated structures of three VacA mutant proteins that all form oligomers but differ in activity. Overall, these studies provide the most detailed analysis of p33 structure to date and also provide a more thorough understanding of how VacA forms oligomers.