Structural, Functional, and Phylogenetic Analyses of the Helicobacter pylori Vacuolating Toxin (VacA)

Abstract

Helicobacter pylori is a gram-negative bacterium that contributes to the pathogenesis of peptic ulcer disease and gastric cancer. A key virulence factor for the organism is vacuolating cytotoxin A (VacA), a pore-forming toxin that causes multiple alterations in human cells. The toxin is secreted by an autotransporter pathway as an 88 kDa protein which can be divided into two structural domains; a 33 kDa domain (p33) responsible for pore formation and a genetically diverse 55 kDa domain (p55) associated with receptor binding. We hypothesize that structural, functional and phylogenetic analyses will allow us to assess the mechanism by which VacA interacts with host cells. Using X-ray crystallography, we found that the VacA m1 p55 domain adopts a β-helix fold, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in β-sheet contacts that result in five β-helix sub-domains and a novel C-terminal domain. The elongated β-helical structure of VacA presents a unique opportunity for studying the pressures of positive and negative selection in a structural context. We have demonstrated that amino acid substitutions in the VacA p55 domain are under strong, diversifying selection. Docking the p55 structure into a 19 Å cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation. We have recently been able to reconstitute this activity from a mixture of p55 and p33 added together in trans. This advance provides a tractable avenue for future studies aimed at obtaining high resolution structural information for VacA p33.