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Low-density lipoprotein receptor-related protein-1 (LRP1) mediates autophagy and apoptosis caused by Helicobacter pylori VacA.

ABSTRACT: In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA receptor for toxin-induced autophagy in the gastric epithelial cell line AZ-521, and show that VacA internalization through binding to LRP1 regulates the autophagic process including generation of LC3-II from LC3-I, which is involved in formation of autophagosomes and autolysosomes. Knockdown of LRP1 and Atg5 inhibited generation of LC3-II as well as cleavage of PARP, a marker of apoptosis, in response to VacA, whereas caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-fmk), and necroptosis inhibitor, Necrostatin-1, did not inhibit VacA-induced autophagy, suggesting that VacA-induced autophagy via LRP1 binding precedes apoptosis. Other VacA receptors such as RPTPα, RPTPβ, and fibronectin did not affect VacA-induced autophagy or apoptosis. Therefore, we propose that the cell surface receptor, LRP1, mediates VacA-induced autophagy and apoptosis.

SUBMITTER: Yahiro K

PROVIDER: S-EPMC3438942 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Endoplasmic reticulum stress contributes to Helicobacter pylori VacA-induced apoptosis.

Project description:Vacuolating cytotoxin A (VacA) is one of the important virulence factors produced by H. pylori. VacA induces apoptotic cell death, which is potentiated by ammonia. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. Our aim was to determine whether endoplasmic reticulum (ER) stress is associated with VacA-induced mitochondrial dysfunction and apoptosis. We found that C/EBP homologous protein (CHOP), a key signaling protein of ER stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with VacA. The effect of VacA on CHOP induction was significantly enhanced by co-incubation with ammonium chloride. Phosphorylation of eukaryotic initiation factor 2 (eIF2)-alpha, which is known to occur downstream of the ER stress sensor PKR-like ER-localized eIF2-alpha kinase (PERK) and to regulate CHOP expression, was also observed following incubation with VacA in the presence of ammonium chloride. Knockdown of CHOP by siRNA resulted in inhibition of VacA-induced apoptosis. Further studies showed that silencing of the PERK gene with siRNA attenuated VacA-mediated phosphorylation of eIF2-alpha, CHOP induction, expression of BH3-only protein Bim and Bax activation, and cell death induced by VacA with ammonium chloride, indicating that ER stress may lead to mitochondrial dysfunction during VacA-induced toxicity. Activation of ER stress and up-regulation of BH3-only proteins were also observed in human H. pylori-infected gastric mucosa. Collectively, this study reveals a possible association between VacA-induced apoptosis in gastric epithelial cells, and activation of ER stress in H. pylori-positive gastric mucosa.

| S-EPMC3862672 | biostudies-literature

Amino-terminal hydrophobic region of Helicobacter pylori vacuolating cytotoxin (VacA) mediates transmembrane protein dimerization.

Project description:Helicobacter pylori VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells. A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences. To examine functional properties of this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W. P. Russ and D. M. Engelman (Proc. Natl. Acad. Sci. USA 96:863-868, 1999) to study transmembrane helix-helix associations in a natural membrane environment. A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of Escherichia coli and mediated protein dimerization. A fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 of VacA was replaced by alanine) also inserted into the inner membrane but dimerized significantly less efficiently than the fusion protein containing the wild-type VacA sequence. Based on these results, we speculate that the wild-type VacA amino-terminal hydrophobic region contributes to oligomerization of the toxin within membranes of eukaryotic cells.

| S-EPMC98002 | biostudies-literature

Expanding allelic diversity of Helicobacter pylori vacA.

Project description:The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of vacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pylori isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA and cagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.

| S-EPMC105169 | biostudies-literature

Expanding Allelic Diversity of Helicobacter pylori vacA

Project description: Not available

| S-EPMC86850 | biostudies-literature

Reconstitution of Helicobacter pylori VacA toxin from purified components.

Project description:Helicobacter pylori VacA is a pore-forming toxin that causes multiple alterations in human cells and contributes to the pathogenesis of peptic ulcer disease and gastric cancer. The toxin is secreted by H. pylori as an 88 kDa monomer (p88) consisting of two domains (p33 and p55). While an X-ray crystal structure for p55 exists and p88 oligomers have been visualized by cryo-electron microscopy, a detailed analysis of p33 has been hindered by an inability to purify this domain in an active form. In this study, we expressed and purified a recombinant form of p33 under denaturing conditions and optimized conditions for the refolding of the soluble protein. We show that refolded p33 can be added to purified p55 in trans to cause vacuolation of HeLa cells and inhibition of IL-2 production by Jurkat cells, effects identical to those produced by the p88 toxin from H. pylori. The p33 protein markedly enhances the cell binding properties of p55. Size exclusion chromatography experiments suggest that p33 and p55 assemble into a complex consistent with the size of a p88 monomer. Electron microscopy of these p33/p55 complexes reveals small rod-shaped structures that can convert to oligomeric flower-shaped structures in the presence of detergent. We propose that the oligomerization observed in these experiments mimics the process by which VacA oligomerizes when in contact with membranes of host cells.

| S-EPMC2910095 | biostudies-literature

Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA.

Project description:The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.

| S-EPMC2374909 | biostudies-literature

Molecular evolution of the Helicobacter pylori vacuolating toxin gene vacA.

Project description:Helicobacter pylori is a genetically diverse organism that is adapted for colonization of the human stomach. All strains contain a gene encoding a secreted, pore-forming toxin known as VacA. Genetic variation at this locus could be under strong selection as H. pylori adapts to the host immune response, colonizes new human hosts, or inhabits different host environments. Here, we analyze 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 positively selected sequence changes in the p55 domain, a central region required for binding of the toxin to host cells. Divergent amino acids map to surface-exposed sites in the p55 crystal structure. Comparative phylogenetic analyses of vacA sequences and housekeeping gene sequences indicate that vacA does not share the same evolutionary history as the core genome. Further, rooting the VacA tree with outgroup sequences from the close relative Helicobacter acinonychis reveals that the ancestry of VacA is different from the African origin that typifies the core genome. Finally, sequence analyses of the virulence determinant CagA reveal three main groups strikingly similar to the three groups of VacA sequences. Taken together, these results indicate that positive selection has shaped the phylogenetic structure of VacA and CagA, and each of these virulence determinants has evolved separately from the core genome.

| S-EPMC2981223 | biostudies-literature

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

Project description:UNLABELLED:Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. MATERIALS AND METHODS:The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. RESULTS:Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient's sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E . co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

| S-EPMC3758054 | biostudies-literature

Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.

Project description:Helicobacter pylori colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pylori strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).

| S-EPMC7240096 | biostudies-literature

Structural analysis of the oligomeric states of Helicobacter pylori VacA toxin.

Project description:Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large "snowflake"-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of ?-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacA?6-27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacA?6-27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacA?301-328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.

| S-EPMC3612943 | biostudies-literature

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