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^-=HCO^3- > 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.  VacA exhibits two characteristics of ClC channels (TC# 2.A.49): an open probability dependent on the molar ratio of permeable ions, and single channel events resolvable as two independent, voltage-dependent transitions (Czajkowsky et al. 2005). The sharing of such peculiar properties by VacA and host ClC channels, together with their similar magnitudes of conductance, ion selectivities, and localization within eukaryotic cells, suggests a novel mechanism of toxin action in which the VacA pore largely mimics the electrophysiological behavior of a host channel, differing only in the membrane potential at which it closes. As a result, VacA can perturb, but not necessarily abolish, the homeostatic ionic imbalance across a membrane and so change cellular physiology without necessarily jeopardizing vitality (Czajkowsky et al. 2005).

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. 

H. pylori VacA enhances the ability of the bacteria to colonize the stomach and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease. The amino acid sequence and structure of VacA are unrelated to corresponding features of other known bacterial toxins. VacA is a pore-forming toxin, and many of its effects on host cells are attributed to formation of channels in intracellular sites. The most extensively studied VacA activity is its capacity to stimulate vacuole formation, but the toxin has many additional effects on host cells (Foegeding et al. 2016). Multiple cell types are susceptible to VacA, including gastric epithelial cells, parietal cells, T cells, and other types of immune cells. 

Cryoelectron microscopy has been used to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers (Zhang et al. 2019). The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed beta-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, short- and/or long-range hydrophobic interactions between protomers across the two layers were found. These structures and other previous observations led to a mechanistic model wherein VacA hexamer correspond to the prepore-forming state, and the N-terminal region of VacA, responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation (Zhang et al. 2019).

The transport reaction catalyzed by VacA is:

Small molecules (in) small molecules (out)