Kinnosuke Yahiro

Mice deficient in protein tyrosine phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter pylori

The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. Here we report that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-ζ or RPTP-β, encoded by Ptprz) do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz−/− mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein–coupled receptor kinase–interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.

Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase β

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-β(1–3)-N-acetylgalactosamine and galactose-β(1–4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPβ/PTPζ); in agreement, p250 reacted with anti-human RPTPβ monoclonal antibody. Immunoprecipitation with anti-human RPTPβ antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPβ bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPβ.

Cutting Edge: VacA, a Vacuolating Cytotoxin of Helicobacter pylori , Directly Activates Mast Cells for Migration and Production of Proinflammatory Cytokines

Mucosal mast cells strategically located at the optimal site interact with invading bacteria. Presence of VacA, the virulent Helicobacter pylori cytotoxin, is correlated with the severity of H. pylori-induced gastritis. To examine the mechanisms of inflammation in H. pylori-induced gastritis, we administered VacA to the mice. Inoculation of VacA resulted in epithelium vacuolization and marked infiltrations of mast cells and mononuclear cells into the mucosal epithelium within 24 h. In an in vitro study using bone marrow-derived mast cells, VacA directly bound and showed a chemotactic activity to the mast cell. In addition, VacA induced bone marrow-derived mast cells to produce proinflammatory cytokines, TNF-α, macrophage-inflammatory protein-1α, IL-1β, IL-6, IL-10, and IL-13 in a dose-dependent manner without causing degranulation. The present study suggests that early activation of mast cells by VacA may be the host early response to clear the bacteria and also may contribute to the pathogenesis of H. pylori-induced gastritis.

Essential domain of receptor tyrosine phosphatase β (RPTPβ) for interaction with Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPβ and RPTPα, RPTPβ was found to be responsible for gastric damage caused by VacA. To define the region of RPTPβ involved in VacA binding, we made mutants of human cDNA RPTPβ-B, a short receptor form of RPTPβ. Immunoprecipitation experiments to assess VacA binding to RPTPβ-B mutants indicated that five residues (QTTQP) at positions 747–751 of the extracellular domain of RPTPβ-B (which is commonly retained in RPTPβ-A, a long form of RPTPβ) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Δ752, which lacks QTTQP, or the double point mutant Δ747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPβ-B and Δ747 (which have QTTQP at 747–751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPβ ligand, on VacA binding to RPTPβ-B or Δ747 was observed, supporting the conclusion that the extracellular region of RPTPβ-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPβ extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747–751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.

Analysis of Helicobacter pylori vacA gene and serum antibodies to VacA in Japan.

Vacuolating cytotoxin, VacA, is one of the most important pathogenetic factors produced by Helicobacter pylori. However, it is not clear whether the diversity in disease outcome may be ascribed to variations in strain and/or to the host responses to virulence factors. In this study, we analyzed the vacA middle region sequence among 65 Japanese isolates to clarify the variation in strain and assayed antibody titer to VacA by ELISA using purified VacA to evaluate the host response to cytotoxin. The nucleotide sequence identities compared among Japanese isolates were 92.8 ± 3.56%, and compared to 88.3 ± 2.89% in tox+ strains reported in GenBank. Positive correlation was found between the antibody titers and the severity of atrophic change of the stomach. In Japan the nucleotide sequences of the vacA middle region were highly homologous and genetically closer to tox+ strains. Antibody titers and host response to cytotoxin may be associated with atrophy of the stomach.

Relationship Between Gastric Ulcer and Helicobacter pylori VacA Detected in Gastric Juice Using Bead-ELISA Method

Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead‐ELISA, to detect VacA in gastric juice.

Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Vacuolating Cytotoxin-Induced Autophagy and Apoptosis During Helicobacter pylori Infection

Abstract Helicobacter pylori –produced vacuolating cytotoxin, VacA, is a major virulence factor, which induces not only vacuole formation but also has other major effects on eukaryotic cells, resulting in apoptotic cell death. Administration of VacA in mice results in severe gastric injury, suggesting that this toxin is an important factor in H. pylori –mediated disease. To understand the disruption of signal transduction by VacA at the early stage, we have identified VacA receptors, receptor-like protein tyrosine phosphatases α and β, on the target cells. We recently identified a novel VacA receptor, low-density lipoprotein receptor-related protein-1 (LRP1), and demonstrated its interaction with VacA. Confocal microscopy revealed that both LRP1 and VacA are partially colocalized with LC3-II in cells after a 12xa0h incubation. Furthermore, we found that knockdown of LRP1 by siRNA significantly suppressed VacA-induced autophagy and apoptosis. Thus, these results indicate that LRP1 is a functional receptor for VacA.