1.C.9 The Vacuolating Cytotoxin (VacA) Family
The bacterium Helicobacter pylori causes chronic superficial gastritis, peptic ulcers and gastric carcinoma in humans. One of the principle virulence factors is the vacuolating cytotoxin VacA, a 90 kDa protein that at neutral pH self-associates into flower shaped dodecameric complexes comprised of two opposing hexamers. In tissue culture cells, VacA causes swelling (vacuolation) of acidic compartments (late endosomes and lysosomes). It engages the mitochondrial fission machinery to induce host cell death (Jain et al., 2011). It has two domains, p33 and p55. p55 is for intracellular toxin activity and assembly of functional oligomeric complexes (Ivie et al., 2008). It acts as an invasive chloride channel tageting mitochondria and causing loss of the mitochondrial membrane potential, mitochondrial fragmentation, formation of reactive oxygen species, autophagy, cell death and gastric cancer (Rassow and Meinecke 2012).
VacA initiates its toxic affect by binding to the cytoplasmic membrane and becoming internalized. It associates with anionic phospholipids in model lipid membranes at pH <5 yielding hexameric membrane-bound complexes. Under these conditions it forms anion selective pores in the bilayers that can be measured electrophysiologically. Thus, low pH and acidic phospholipids probably induce channel-formation by inducing a conformational change in the oligomeric complex that opens the channel. This process is presumably related to its vacuolating cytotoxic activity. Ion selectivity was shown to be Cl-=HCO3- > pyruvate > gluconate >K =Li =Ba2 > NH4 .
Some parallels have been noted between the function of VacA and various A-B type toxins such as diphtheria toxin (DT; TC #1.C.7) and botulinum toxin (in the BTT family; TC #1.C.8). However, no significant sequence similarity with DT, members of the BTT family, or any other well-characterized toxin is observed. Their mechanisms of pore formation may be very different.
The x-ray structure of the H pylori VacA and p55 domain has been solved by x-ray diffraction at 2.4 Å resolution. This toxin is secreted by an autotransporter pathway, contributing to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. The structure is predominantly a right-handed parallel beta-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in beta-sheet contacts that result in five beta-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allowed identification of structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-A cryo-EM map of a VacA dodecamer allowed proposal of a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation (Gangwer et al., 2007).
Helicobacter pylori is adapted for colonization of the human stomach. All strains contain VacA. Genetic variation at this locus could allow adaptation to the host immune response. Gangwer et al. (2010) analyzed the molecular evolution of VacA. Phylogenetic reconstructions indicate the subdivision of VacA sequences into three main groups with distinct geographic distributions. Divergence of the three groups is principally due to sequence changes in surface-exposed sites in the p55 crystal structure domain, a central region required for binding of the toxin to host cells.
The bipartite vacuolating cytotoxin A, VacA, of Helicobacter pylori enters host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). VacA forms large multimeric pores composed of both subunits in membranes. A major target organelle of VacA is the mitochondrion. Foo et al., 2010 showed that both subunits are imported into mitochondria. The p33 subunit integrally associates with the mitochondrial inner membrane, and both subunits are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they reassemble to form a pore in the inner mitochondrial membrane.
The transport reaction catalyzed by VacA is thus:
small molecules (in) small molecules (out).