Intoxication strategy of Helicobacter pylori VacA toxin

VacA toxin from the cancer-inducing bacterium Helicobacter pylori is currently classified as a pore-forming toxin but is also considered a multifunctional toxin, apparently causing many pleiotropic cell effects. However, an increasing body of evidence suggests that VacA could be the prototype of a new class of monofunctional A-B toxins in which the A subunit exhibits pore-forming instead of enzymatic activity. Thus, VacA may use a peculiar mechanism of action, allowing it to intoxicate the human stomach. By combining the action of a cell-binding domain, a specific intracellular trafficking pathway and a novel mitochondrion-targeting sequence, the VacA pore-forming domain is selectively delivered to the inner mitochondrial membrane to efficiently kill target epithelial cells by apoptosis.     

The influence of bacterial toxins on the carcinogenesis

Bacterial infections may constitute an important risk factor of developing cancer disease. Molecular mechanisms by which bacteria contribute to cancer are extremely complex and still remain not fully understood. So far, it is generally accepted that Helicobacter pylori infections are associated with induction of gastric adenocarcinoma and MALT lymphoma. Two H. pylori toxins which modulate many cellular functions are VacA and CagA. So far, CagA is the only one known bacterial oncoprotein. However, many other bacteria produce toxins or effector proteins perturbing host cell homeostasis or/and evoking chronic inflammation. Both processes may be associated with tumour formation. Bacterial toxins which interfere, with various host signal transduction pathways, deregulate processes of cell division, proliferation and differentiation and modulate apoptosis. Some toxins cause even direct DNA damage. This review discuss the potential links between action of bacterial toxins and cancer.     

幽门螺杆菌vacA基因的克隆和序列分析

空泡毒素是幽门螺杆菌产生的已知的唯一蛋白毒素,该毒素与感染者胃肠上皮务和溃疡形成密切相关,同时也是幽门螺杆菌免疫预防和免疫治疗的重要候选组分。从幽门螺杆菌NCTC11637染色体DNA中经PCR方法获得了2.9kb的该基因成熟肽全长序列,将该基因克隆至载体pET22b ,经PCR扩增和酶切鉴定后序列分析表明,该基因与已知序列完全一致。     

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.     

Functional properties of the p33 and p55 domains of the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.     

Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism

Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.     

A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.     

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling

To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.     

Prevalence of CagA, VacA Antibodies in Symptomatic and Asymptomatic Children with Helicobacter pylori Infection

Background. Limited data are available on the prevalence of CagA and VacA Helicobacter pylori antibodies in children. The aim of this study was to investigate the antibody prevalence to the H. pylori virulence factors CagA and VacA in symptomatic and asymptomatic children with H. pylori infection and to correlate these antibodies with the severity of gastric inflammation or density of H. pylori organisms in the gastric mucosa.
Materials and Methods. Twenty-three symptomatic children and 132 asymptomatic children with positive H. pylori serology participated in this study. Anti– H. pylori IgG antibody and CagA or VacA H. pylori antibodies were measured by enzyme immunoassay (HM-CAP; sensitivity and specificity> 90%) and Western immunoblot (Helicoblot 2.0) methods, respectively. Gastric inflammation and H. pylori density were graded histologically using the revised Sydney criteria.
Results. The prevalence of CagA and VacA antibodies were 69% and 35% in symptomatic children and 54% and 52% in asymptomatic children, respectively. Multiple regression analysis showed a correlation between CagA antibody and the severity of gastritis but no correlation with other histological features, including the number of neutrophils or lymphoid follicles. Neither antibody correlated with the degree of bacterial density in the gastric mucosa.
Conclusion. CagA and VacA H. pylori antibodies are common in the pediatric population. The combined CagA/VacA antibodies correlated weakly with the degree of mucosal inflammation.     

PKC-dependent endocytosis of the Helicobacter pylori vacuolating cytotoxin in primary T lymphocytes

Gastric infection by Helicobacter pylori (Hp) is associated with development of gastritis, ulcerations and gastric adenocarcinoma. Production and secretion of the vacuolating cytotoxin (VacA) is an essential Hp virulence factor. VacA is a multifunctional toxin, which exerts immunosuppressive effects on human T lymphocytes via inhibition of cell proliferation and Interleukin-2 (IL-2) signalling. This latter effect of VacA is dependent on the β2-integrin subunit CD18, acting as a receptor for intracellular uptake of VacA. In this study, we investigated the mechanism of endocytosis of VacA into primary human T lymphocytes. A screen with chemical inhibitors for different sets of kinases identified Ser/Thr kinases of the protein kinase C (PKC) family as crucial. Specific inhibitory peptides blocking PKCη or PKCζ-phosphorylating activity, but not PKCα/β specific peptides, resulted in a strong reduction or complete block of VacA uptake. Thus the phosphorylating activity of PKCη and PKCζ is essential for the induction of VacA endocytosis. Furthermore, mimicking of a possible PKC-mediated threonine (T(758)) phosphorylation of the CD18 cytoplasmic tail in resting primary T cells induced VacA endocytosis via activation of the small GTPases Cdc42 and Rac-1. We conclude that VacA is endocytosed into primary T cells via a clathrin-independent pathway.     

Clustering and redistribution of late endocytic compartments in response to Helicobacter pylori vacuolating toxin

Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form anion-selective membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.     

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.     

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.     

Beta2 integrin mediates entry of a bacterial toxin into T lymphocytes

Helicobacter pylori secretes a pore-forming toxin, VacA, that can cause numerous alterations in gastric epithelial cells and T lymphocytes. In this issue of Cell Host & Microbe, Sewald and colleagues report that beta2 integrin (CD18) mediates entry of VacA into human T cells. Downregulation of T cell responses by VacA may allow H. pylori to evade the adaptive immune response and establish persistent infection.     

抗重组VacA IgY体内抗幽门螺杆菌感染的实验研究

目的在构建H.pylori的基因工程菌pQE30-v-DH5a的基础上,诱导表达VacA重组蛋白,以此为抗原,制备抗VacA的蛋黄抗体（VacA IgY）。通过小鼠口服试验,证实VacA IgY治疗H.pylori感染的作用,为进一步制备抗H．pylori感染的IgY制剂提供实验依据。方法用重组H.pylori VacA蛋白免疫母鸡,水稀释结合氯仿有机沉淀法提取IgY,ELISA法测定其针对VacA的效价。建立H.pylori感染的Balb／c小鼠动物模型,治疗组在小鼠灌喂菌液后灌喂不同剂量的VacA IgY。以H.pylori培养和病理切片观察胃黏膜H.pylori定植和炎症反应程度。结果制备了高效价的IgY（1：12800）。动物实验阳性对照组H.pylori的总感染率为70．4％,12周后的感染率为88．9％。治疗组的感染率与同期阳性对照组相似,胃黏膜的炎症反应程度比阳性对照组弱,随IgY剂量的增加,炎症减弱明显,IgY剂量为4mg／ml时,能达到较理想的治疗效果。结论成功制备了高效价的特异性VacA IgV,小鼠体内实验证实了口服VacA IgY具有治疗H.pylori感染的作用,可用于制备口服制剂。     

Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets

Helicobacter pylori are Gram-negative bacteria that persistently colonize the human gastric mucosa despite the recruitment of immune cells. The H. pylori vacuolating cytotoxin (VacA) recently has been shown to inhibit stimulation-induced proliferation of primary human CD4(+) T cells. In this study, we investigated effects of VacA on the proliferation of various other types of primary human immune cells. Intoxication of PBMC with VacA inhibited the stimulation-induced proliferation of CD4(+) T cells, CD8(+) T cells, and B cells. VacA also inhibited the proliferation of purified primary human CD4(+) T cells that were stimulated by dendritic cells. VacA inhibited both T cell-induced and PMA/anti-IgM-induced proliferation of purified B cells. Intoxication with VacA did not alter the magnitude of calcium flux that occurred upon stimulation of CD4(+) T cells or B cells, indicating that VacA does not alter early signaling events required for activation and proliferation. VacA reduced the mitochondrial membrane potential of CD4(+) T cells, but did not reduce the mitochondrial membrane potential of B cells. We propose that the immunomodulatory actions of VacA on T and B lymphocytes, the major effectors of the adaptive immune response, may contribute to the ability of H. pylori to establish a persistent infection in the human gastric mucosa.