CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin‐associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty‐eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive‐ or negative‐CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.     

CagA phosphorylation-dependent MMP-9 expression in gastric epithelial cells

Background: Infection of cagA‐positive Helicobacter pylori is associated with increased expression of MMPs in gastric epithelial cells. The role of phosphorylated CagA in the induction of MMP‐9, a protease‐degrading basement membrane, in gastric epithelial cells has not been clearly defined yet. The aim of this study is to analyze whether the presence of CagA and its phosphorylation status play a role in increased expression of MMP‐9 in gastric epithelial cells. Materials and Methods: Induction of MMP‐9 secretion was analyzed in gastric epithelial AGS cells harboring CagA with or without EPIYA motif, which is injected by H. pylori or ectopically expressed. In addition, signaling pathways involved in the CagA‐dependent MMP‐9 production have been studied. Results: The 147C strain of H. pylori expressing tyrosine‐phosphorylated CagA (EPIYA present) induced higher MMP‐9 secretion by AGS cells than the 147A strain expressing non‐tyrosine‐phosphorylated CagA (EPIYA absent). In addition, in bacteria‐free CagA‐inducible AGS cells, expression of wild‐type CagA induced more MMP‐9 secretion than phosphorylation‐resistant CagA. Inhibition of CagA phosphorylation by the Src family kinase inhibitor PP1 downregulated CagA‐mediated MMP‐9 secretion. Knockdown of SHP‐2 phosphatase dramatically reduced MMP‐9 secretion. ERK inhibitors, PD98059 and U0126, and NF‐κB pathway inhibitors, sulfasalazine and N‐acetyl‐l ‐cysteine, also inhibited MMP‐9 expression. Conclusion: These results support a model whereby the EPIYA motif of CagA is phosphorylated by Src family kinases in gastric epithelial cells, which initiates activation of SHP‐2. In addition, they suggest that the resultant activation of ERK pathway along with CagA‐dependent NF‐κB activation is critical for the induction of MMP‐9 secretion.     

Haem oxygenase‐1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

The cytotoxin‐associated gene A protein (CagA) plays a pivotal role in the aetiology of Helicobacter pylori‐associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c‐Src. We previously reported that the enzyme haem oxygenase‐1 (HO‐1) inhibits IL‐8 secretion by H. pylori‐infected cells. However, the cellular mechanism by which HO‐1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and haemin, two inducers of HO‐1, decrease the level of phosphorylated CagA (p‐CagA) in H. pylori‐infected gastric epithelial cells and this is blocked by either pharmacological inhibition of HO‐1 or siRNA knockdown of hmox‐1. Moreover, forced expression of HO‐1 by transfection of a plasmid expressing hmox‐1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori‐induced c‐Src phosphorylation/activation by HO‐1.Consequently, H. pylori‐induced cytoskeletal rearrangements and activation of the pro‐inflammatory response mediated by p‐CagA are inhibited in HO‐1‐expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells.     

Expression of CEACAM1 or CEACAM5 in AZ‐521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin‐associated genes pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c‐Src and c‐Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ‐521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA‐dependent fashion. In contrast, we discovered that AZ‐521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin‐β₁ or carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ‐521 cells readily express integrin‐β₁, but overexpression of integrin‐β₁ constructs did not restore the T4SS defect. We further show that AZ‐521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ‐521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ‐521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori‐infected AGS cells. Our results suggest the existence of an integrin‐β₁‐ and CEACAM1‐ or CEACAM5‐dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.     

Helicobacter pylori CagA: Analysis of Sequence Diversity in Relation to Phosphorylation Motifs and Implications for the Role of CagA as a Virulence Factor

CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y-122 in CagA is homologous with a pair of PDZ domains which are common in signal transducing proteins, particularly tyrosine phosphatases. This provides a theoretical link between CagA and many of the observed responses of host cells to H. pylori. In summary, not all CagA proteins are equal in their potential for initiating host cell responses via signal transduction pathways. The degree of functional diversity of this protein depends upon which phosphorylation motifs are critical to the biological activity of CagA.     

Subversion of host kinases: a key network in cellular signaling hijacked by Helicobacter pylori CagA

Helicobacter pylori is a paradigm of persistent pathogens and major risk factor for developing severe diseases including adenocarcinoma in the human stomach. An important bacterial factor linked to gastric disease progression is the cag pathogenicity island‐encoded type‐IV secretion system (T4SS) effector protein CagA. Translocated CagA undergoes tyrosine phosphorylation at EPIYA‐motifs and then activates or inactivates multiple host signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent fashion. In this way, intracellular CagA acts as a ‘masterkey’ or ‘picklock’, which evolved during evolution to hijack key host cell signal transduction functions. Crucial targets of CagA represent a variety of serine/threonine and tyrosine kinases, which control major checkpoints of eukaryotic signaling. Here we review the signal transmission by translocated CagA on multiple receptor kinases (c‐Met and EGFR) and non‐receptor kinases (Src, Abl, Csk, aPKC, Par1, PI3K, Akt, FAK, GSK‐3, JAK, PAK1, PAK2 and MAP kinases), manipulating a selection of fundamental processes in the human gastric epithelium such as cell adhesion, polarity, proliferation, motility, receptor endocytosis, cytoskeletal rearrangements, apoptosis, inflammation and cell cycle progression. This enormous complexity generates a highly remarkable and puzzling scenario during H. pylori infection. The contribution of these signaling pathways to bacterial survival, persistence and gastric pathogenesis is discussed.     

Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacA

Helicobacter pylori is the causative agent of gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent strains carrying both the cag pathogenicity island ( cag PAI) and the vacuolating cytotoxin VacA are key players in disease development. The ca gPAI encodes a type IV secretion system (T4SS) which forms a pilus for injection of the CagA protein into gastric epithelial cells. Injected CagA undergoes tyrosine phosphorylation and induces actin-cytoskeletal rearrangements involved in host cell scattering and elongation. We show here that the CagA-induced responses can be inhibited in strains expressing highly active VacA. Further investigations revealed that VacA does not interfere with known activities of phosphorylated CagA such as inactivation of Src kinase and cortactin dephosphorylation. Instead, we demonstrate that VacA exhibits inactivating activities on the epidermal growth factor receptor EGFR and HER2/Neu, and subsequently Erk1/2 MAP kinase which are important for cell scattering and elongation. Inactivation of vacA gene, downregulation of the VacA receptor RPTP-α, addition of EGF or expression of constitutive-active MEK1 kinase restored the capability of H. pylori to induce the latter phenotypes. These data demonstrate that VacA can downregulate CagA's effects on epithelial cells, a novel molecular mechanism showing how H. pylori can avoid excessive cellular damage.     

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. Materials and Methods. Forty‐eight H. pylori‐positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori‐negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the antibody to VacA, were used in this bead‐ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentrations of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relationship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead‐ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage.     

Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin

Background. Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori‐vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori‐induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo. Materials and Methods. Effects of polyphenols on VacA were quantified by the inhibition of: 1, vacuolation; 2, VacA binding to AZ‐521 or G401 cells or its receptors; 3, VacA internalization. Effects of hop bract extract (HBT) containing high molecular weight polymerized catechin on VacA in vivo were investigated by quantifying gastric damage after oral administration of toxins to mice. Results. HBT had the strongest inhibitory activity among the polyphenols investigated. HBT inhibited, in a concentration‐dependent manner: 1, VacA binding to its receptors, RPTPα and RPTPβ; 2, VacA uptake; 3, VacA‐induced vacuolation in susceptible cells. In addition, oral administration of HBT with VacA to mice reduced VacA‐induced gastric damage at 48 hours. In vitro, VacA formed a complex with HBT. Conclusions. HBT may suppress the development of inflammation and ulceration caused by H. pylori VacA, suggesting that HBT may be useful as a new type of therapeutic agent for the prevention of gastric ulcer and inflammation caused by VacA.     

Dynamics of the Cag‐type IV secretion system of Helicobacter pylori as studied by bacterial co‐infections

Many pathogenic Gram‐negative bacteria possess type IV secretion systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major aetiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the cag‐T4SS to inject the cytotoxin associated Antigen (CagA) into gastric epithelial cells. This results in deregulation of major signalling cascades, actin‐cytoskeletal rearrangements and eventually gastric cancer. We show here that a pre‐infection with live H. pylori has a dose‐dependent negative effect on the CagA translocation efficiency of a later infecting strain. This effect of the ‘first’ strain was independent of any of its T4SS, the vacuolating cytotoxin (VacA) or flagella. Other bacterial pathogens, e.g. pathogenic Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, or commensal bacteria, such as lactobacilli, were unable to interfere with H. pylori's CagA translocation capacity in the same way. This interference was independent of the β1 integrin receptor availability for H. pylori, but certain H. pylori outer membrane proteins, such as HopI, HopQ or AlpAB, were essential for the effect. We suggest that the specific interference mechanism induced by H. pylori represents a cellularresponse to restrict and control CagA translocation into a host cell to control the cellular damage.     

Clinical application of 20 MHz endosonography and anti-Helicobacter pylori immunoblots to predict regression of low-grade gastric MALToma by H. pylori eradication

Aim. We tested whether serial 20 MHz endosonography (EUS) and anti‐Helicobacter pylori immunoblots can predict the complete regression of gastric MALToma by H. pylori eradication. Methods. The serums of 17 MALToma patients, including 15 with low grade and two with high grade, were collected before therapy. Fifteen patients with low‐grade MALToma and 18 nonMALToma patients, all infected with H. pylori, have been followed with serum sampling, endoscopy, and EUS on enrollment, on the 2nd, 6th, and 12th months after anti‐H. pylori therapy. All sera were tested for anti‐H. pylori immunoblots, including 19.5, 26.5, 30, 35, 89, 116 KDa (CagA), FldA. The DNAs were extracted serially from the biopsy of MALToma patients before and after therapy to perform polymerase chain reaction (PCR) for the immunoglobulin heavy‐chain gene monoclonality. Results. MALToma patients had higher prevalence rates of anti‐FldA protein, 19.5 and 30 KDa antibodies of H. pylori (p < 0.01). After H. pylori eradication, MALToma patients had negative seroconversion of 19.5, 26.5, 30, and 35 KDa antibodies (p < 0.05), but not in CagA and FldA. The PCR monoclonality occurred in 80% (12/15) of the MALToma patients before therapy, but did not correlate with any seroconversion of anti‐H. pylori immunoblots after therapy (p > 0.05). Complete regression of MALToma was observed in 73.3% (11/15) of patients. Evaluation with 20 MHz EUS, for the initial tumor depth and its normalization on the 6th month had 90.9% sensitivity and 100% specificity to predict the complete regression. Discussion. The negative seroconversions of the smaller‐molecular‐weight proteins, but not CagA and FldA, correlate with regression of MALToma by H. pylori eradication. 20 MHz EUS can effectively predict the therapeutic response of MALToma.     

Review: Pathogenesis of Helicobacter pylori infection

In this review, we shall focus on the last year progression understanding the pathogenesis of Helicobacter pylori infection in the light of recent data related to adaptation of H pylori to the harsh acidic environment in the stomach, colonization of gastric mucosa via interaction with mucin 5 (MUC5AC) and other host cell receptors, the ability to form biofilm, interference with the host metabolic pathways, and induction of neuroimmune cross‐talk as well as downregulation of gastric barrier homeostasis and its consequences for the disease development. The role of the membrane vesicles of these bacteria has been emphasized as an important source of virulence factors. Furthermore, we shall describe molecular and functional studies on new aspects of VacA and CagA virulence, including the role of urease in the upregulation of VacA toxicity, an epithelial‐mesenchymal transition mediated by CagA, and the role of interaction of HopQ adhesin with carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) in CagA translocation into the host cells by the type IV secretion system (T4SS). The role of molecular mimicry between a common sequence (ATVLA) of H pylori heat shock protein (Hsp) B and human Hsp60 in the induction of potentially autoreactive antibodies is discussed. All these new data illustrate further progress in understanding H pylori pathogenicity and facilitate the search for new therapeutic targets as well as development of immunoprophylaxis methods based on new chimeric UreB and HpA proteins.     

The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key Hypothesis

Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type‐IV secretion system. Injected CagA becomes tyrosine‐phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high‐resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence‐associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ‘master key’ that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin‐cytoskeletal rearrangements and the disruption of cell‐to‐cell junctions as well as proliferative, pro‐inflammatory and anti‐apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.     

Antral nodularity and positive CagA serology are distinct and relevant markers of severe gastric inflammation in children with Helicobacter pylori infection

Background. The aim of this study was to assess whether the endoscopic finding of antral nodularity and serum IgG antibodies to CagA are associated with higher grades of gastric inflammation. Materials and methods. The comprehensive data of two previously published trials were reanalysed. One hundred and fifty‐three children (median age 9.5 years) who underwent gastroscopy were included. Biopsy specimens from the antrum and corpus were taken to assess Helicobacter pylori status, gastritis score and lymphoid follicles. During endoscopy, antral nodularity was noted. Serum samples were assayed for IgG antibodies to CagA. Results. The presence of antral nodularity (nod+) and positive CagA serology (CagA+) were each found in 32 of the 77 (41.5%) children who had evidence of H. pylori infection. Crosstabulation showed that 20 children (26%) were nod+/CagA+, 12 (15.5%) nod+/CagA?, 12 (15.5%) nod‐/CagA+ and 33 (43%) nod?/CagA?. Gastritis score was significantly lower in nod?/CagA?children than in nod+/CagA? (p = .004), nod?/CagA+ (p = .002) and nod+/CagA+ (p < .001), both in the antrum and corpus. Completely normal gastric histology was only found in the nod?/CagA?subgroup of H. pylori‐infected children (eight of 33, 24%). Regression analysis showed that antral nodularity and positive CagA serology were related to severe gastric inflammation independently of each other and age. Separate analysis showed that inflammation (p < .001), activity (p < .001) and H. pylori density (p = .002) scores were significantly lower in nod?/CagA?children compared with nod+/CagA+ children. The number of lymphoid follicles in the gastric mucosa was related to antral nodularity (p = .003) and positive CagA serology (p = .043), independently of each other. Conclusions. Antral nodularity and positive CagA serology are distinct and relevant markers of severe gastric inflammation in children with H. pylori infection. The lack of both findings in the same child reflects low‐grade or no gastritis.     

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.     

A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.     

NOD1 is required for Helicobacter pylori induction of IL‐33 responses in gastric epithelial cells

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL‐33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL‐33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide‐Binding Oligomerisation Domain‐Containing Protein 1 (NOD1) and its adaptor protein receptor‐interacting serine–threonine Kinase 2, to promote production of both full‐length and processed IL‐33 in gastric epithelial cells. Furthermore, IL‐33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1^+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL‐33 and splenic IL‐13 responses, but decreased IFN‐γ responses, when compared with Nod1^?/? animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL‐33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.