The VacA toxin secreted by Helicobacter pylori 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 classified as 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. Multiple cell types are susceptible to VacA, including gastric epithelial cells, parietal cells, T cells, and other types of immune cells. This review focuses on the wide range of VacA actions that are detectable in vitro, as well as actions of VacA in vivo that are relevant for H. pylori colonization of the stomach and development of gastric disease.

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS. Helicobacter pylori persistently colonizes the stomach in approximately half of the human population. People who are infected with H. pylori strains harboring the cag pathogenicity island (PAI) have an increased risk of developing gastric cancer. The cag PAI encodes a type IV secretion system (T4SS) that is utilized by the bacteria to inject the bacterial oncoprotein CagA into gastric epithelial cells. Related T4SSs found in several other bacteria have been studied in detail, but thus far there has been very little study of the H. pylori cag T4SS. Here, we utilized a mass spectrometry-based approach to reveal co-purification of three constituents of the H. pylori T4SS (CagH, CagI, and CagL) that lack homology to components of T4SSs in other bacterial species. These proteins are essential for CagA translocation into host cells, and scanning electron microscope studies reveal that the proteins are involved in the formation of pili at the bacterial-host cell interface. A conserved C-terminal motif present in CagH, CagI, and CagL is essential for functionality of the T4SS. This study highlights the important role played by unique constituents of the H. pylori cag T4SS, and illustrates the marked variation that exists among bacterial T4SSs.

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G¹⁴ XXXG¹⁸ motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.

Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-Å crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel β-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 β-sheet contacts that result in five β-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, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of 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.

The phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a short DNA element. This element (switch) acts in cis to control transcription of fimA, the major fimbrial subunit gene. Thus, fimA is transcribed when the switch is in one orientation (the on orientation) but not the other (the off orientation). The fim inversion requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only), as well as integration host factor, and is also influenced by the abundant DNA-binding protein H-NS. Here we report that an additional gene, lrp, a factor known to influence the expression of both Pap and K99 fimbriae, is also required for normal activity of the fim switch. The frequencies of both fimB-promoted and fimE-promoted inversions, and consequently the phase variation of type 1 fimbriation, are lower in lrp mutants. Lrp affects slightly the transcription of both fimB (which is increased) and fimE (which is decreased). We believe that these alterations in fimB and fimE transcription alone are unlikely to account for the sharp reduction in switching found in lrp mutants.