The functional status of the Helicobacter pylori sabB adhesin gene as a putative marker for disease outcome

Background. Helicobacter pylori factors that contribute to disease outcome are largely unknown, but intimate contact with host cells mediated by outer membrane proteins is thought to play an important role. Expression of the outer membrane proteins OipA, HopZ, SabA, and SabB is regulated by phase‐variable dinucleotide repeats in the coding regions of the respective genes. We have evaluated the correlation between the expression status of these four genes and disease outcome of H. pylori infection in a Dutch patient population. Materials and Methods. H. pylori strains, isolated from 96 Dutch patients with gastritis (n = 29), duodenal ulcer (n = 28), gastric ulcer (n = 21), gastric carcinoma (n = 9), and lymphoma (n = 9), were analyzed for the ‘on/off’ expression status of the H. pylori genes oipA, hopZ, sabA, and sabB by direct DNA sequence analysis of amplified fragments. Results. The off‐status of sabB was significantly associated with duodenal ulcer (p = .036), but not with gastric ulcer. In contrast, the expression status of oipA, hopZ, and sabA did not correlate with disease outcome. Furthermore, lymphoma strains appeared to express a significantly smaller amount of putative adhesins when compared to gastritis, gastric ulcer, duodenal ulcer and gastric carcinoma strains (p < .02 for all groups tested). Conclusion. The off‐status of sabB was found to be associated with duodenal ulcer disease, and thus represents a putative marker for disease outcome. Assuming that SabB is involved in bacterial adhesion, this association suggests that adherent H. pylori are more prone to elimination by the host immune system.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

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.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

A Potential Association between Helicobacter pylori CagA EPIYA and Multimerization Motifs with Cytokeratin 18 Cleavage Rate during Early Apoptosis

Background and Aims: Helicobacter pylori is a highly diverse pathogen, which encounters epithelial cells as the initial defense barrier during its lifelong infection. The structure of epithelial cells can be disrupted through cleavage of microfilaments. Cytokeratin 18 (CK18) is an intermediate filament, the cleavage of which is considered an early event during apoptosis following activation of effector caspases. Methods: Helicobacter pylori strains were isolated from 76 dyspeptic patients. cagA 3’ variable region and CagA protein status were analyzed by PCR and western blotting, respectively. Eight hours post‐co‐culture of AGS cells with different H. pylori strains, flow cytometric analysis was performed using M30 monoclonal antibody specific to CK18 cleavage‐induced neo‐epitope. Results: Higher rates of CK18 cleavage were detected during co‐culture of AGS cells with H. pylori strains bearing greater numbers of cagA EPIYA‐C and multimerization (CM) motifs. On the other hand, H. pylori strains with greater numbers of EPIYA‐B relative to EPIYA‐C demonstrated a decrease in CK18 cleavage rate. Thus, H. pylori‐mediated cleavage of CK18 appeared proportional to the number of CagA EPIYA‐C and CM motifs, which seemed to be downplayed in the presence of EPIYA‐B motifs. Conclusions: Our observation associating the heterogeneity of cagA variants with the potential of H. pylori strains in the induction of CK18 cleavage as an early indication of apoptosis in gastric epithelial cells supports the fact that apoptosis may be a type‐specific trait. However, additional cagA‐targeted experiments are required to clearly identify the role of EPIYA and CM motifs in apoptosis and/or the responsible effector molecules.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

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.     

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.     

p27(kip1) upregulated by hnRNPC1/2 antagonizes CagA (a virulence factor of Helicobacter pylori)-mediated pathogenesis

Background and Aims: Infection by Helicobacter pylori is one of the major contributing factors of chronic active gastritis and peptic ulcer and is closely associated with the occurrence and progression of gastric cancer. CagA protein is a major virulence factor of H. pylori that interacts with SHP‐2, a true oncogene, to interfere with cellular signaling pathways; CagA also plays a crucial role in promoting the carcinogenesis of gastric epithelial cells. However, currently, the molecular mechanisms of gastric epithelial cells that antagonize CagA pathogenesis remain inconclusive. Methods: We showed that AGS gastric cancer cells transfected with CagA exhibited the inhibition of proliferation and increased activity of caspase 3/7 using two‐dimensional gel electrophoresis and secondary mass spectrometry (MS/MS). Results: It was found that the AGS gastric cancer cells stably expressing CagA displayed significantly increased the expression of 16 proteins, including hnRNPC1/2. Further analysis revealed that hnRNPC1/2 significantly boosted the expression of the p27^kip1 protein. Conclusion: Our data suggested that hnRNPC1/2 upregulates p27^kip1 expression and the subsequent suppression of cell proliferation and induction of apoptosis, thereby providing an important mechanism whereby gastric epithelial cells antagonize CagA‐mediated pathogenesis.     

Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis

Background and Aims. H. pylori infection results in an increased epithelial apoptosis in gastritis and duodenal ulcer patients. We investigated the role and type of activation of caspases in H. pylori‐induced apoptosis in gastric epithelial cells. Methods. Differentiated human gastric cancer cells (AGS) and human gastric mucous cell primary cultures were incubated with H. pylori for 0.5–24 hours in RPMI 1640 medium, and the effects on cell viability, epithelial apoptosis, and activity of caspases were monitored. Apoptosis was analyzed by detection of DNA‐fragments by Hoechst stain®, DNA‐laddering, and Histone‐ELISA. Activities of caspases were determined in fluorogenic assays and by Western blotting. Cleavage of BID and release of cytochrome c were analyzed by Western blot. Significance of caspase activation was investigated by preincubation of gastric epithelial cells with cell permeable specific caspase inhibitors. Results. Incubation of gastric epithelial cells with H. pylori caused a time and concentration dependent induction of DNA fragmentation (3‐fold increase), cleavage of BID, release of cytochrome c and a concomittant sequential activation of caspase‐9 (4‐fold), caspase‐8 (2‐fold), caspase‐6 (2‐fold), and caspase‐3 (6‐fold). No effects on caspase‐1 and ‐7 were observed. Activation of caspases preceded the induction of DNA fragmentation. Apoptosis could be inhibited by prior incubation with the inhibitors of caspase‐3, ‐8, and ‐9, but not with that of caspase‐1. Conclusions. Activation of certain caspases and activation of the mitochondrial apoptotic pathway are essential for H. pylori induced apoptosis in gastric epithelial cells.     

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.     

Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo

Background. Helicobacter pylori CagA is injected into the host cell and tyrosine‐phosphorylated. We examined tyrosine‐phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. Methods. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine‐phosphorylation sites were determined using quadropolar time‐of‐flight (Q‐TOF) mass spectrometry analysis. Specific anti‐pY CagA polyclonal and anti‐CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Results. Mass spectrometry identified five crucial tyrosine‐phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)‐infected AGS cells. CagA protein also had an immuno‐receptor tyrosine‐based activation motif (ITAM)‐like amino acid sequences in the 3′ region of the cagA, E PIY ATI x₂₇EIY ATI , which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX‐100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine‐phosphorylated GIT1/Cat1 (G protein‐coupled receptor kinase‐interactor 1/Cool‐associated tyrosine‐phosphorylated 1), substrate molecules of receptor type protein‐tyrosine phosphatase (RPTPζ/β), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA‐induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine‐phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. Conclusion. These findings suggest an important role for CagA containing ITAM‐like sequences in the pathogenesis of H. pylori‐related disease.     

Fasting gastric pH of Japanese subjects stratified by IgG concentration against Helicobacter pylori and pepsinogen status

Background: The clinical significance of Helicobacter pylori antibody titer has been controversial, and the association between the extent of gastric atrophy or acid secretion and H. pylori antibody concentration has not been elucidated. Materials and Methods: Serum pepsinogen, H. pylori antibody concentration, and fasting gastric pH (as an indicator of acid secretion) were measured in 231 patients undergoing upper gastrointestinal endoscopy. “Atrophic” pepsinogen was defined as pepsinogen‐I < 70 ng/mL and pepsinogen‐I/II ratio <3. Other levels of pepsinogen were defined as “normal”. Fasting gastric pH was analyzed in subjects stratified by pepsinogen level and by H. pylori antibody concentration. Results: Helicobacter pylori antibody concentration showed no significant relationship with fasting gastric pH when all subjects were analyzed together. In H. pylori‐seronegative subjects, fasting gastric pH was within the normal range, irrespective of the extent of mucosal atrophy. In H. pylori‐seropositive subjects, H. pylori antibody concentration was positively correlated with fasting gastric pH in subjects with “normal” pepsinogen, but inversely correlated in those with “atrophic” pepsinogen. Particularly in subjects with low H. pylori antibody concentration and atrophic mucosa, a group reportedly at high risk of noncardia cancer, the most impaired acid secretion was shown among subjects with atrophic mucosa. Conclusions: The relationship between acid secretion and H. pylori antibody concentration differs depending on the presence of mucosal atrophy. Our findings provide a possible rationalization for measuring both serum pepsinogen levels and H. pylori antibody concentration in gastric cancer screening.     

Characterization of Helicobacter pylori VacA‐containing vacuoles (VCVs), VacA intracellular trafficking and interference with calcium signalling in T lymphocytes

The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases such as gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion‐selective channels in the endosome membrane that cause the compartment to swell, but the composition and purpose of the resulting VacA‐containing vacuoles (VCVs) are still unknown. VacA exerts influence on the host immune response in various ways, including inhibition of T‐cell activation and proliferation and suppression of the host immune response. In this study, for the first time the composition of VCVs from T cells was comprehensively analysed to investigate VCV function. VCVs were successfully isolated via immunomagnetic separation, and the purified vacuoles were analysed by mass spectrometry. We detected a set of 122 VCV‐specific proteins implicated among others in immune response, cell death and cellular signalling processes, all of which VacA is known to influence. One of the individual proteins studied further was stromal interaction molecule (STIM1), a calcium sensor residing in the endoplasmic reticulum (ER) that is important in store‐operated calcium entry. Live cell imaging microscopy data demonstrated colocalization of VacA with STIM1 in the ER and indicated that VacA may interfere with the movement of STIM1 towards the plasma membrane‐localized calcium release activated calcium channel protein ORAI1 in response to Ca²⁺ store depletion. Furthermore, VacA inhibited the increase of cytosolic‐free Ca²⁺ in the Jurkat E6‐1 T‐cell line and human CD4⁺ T cells. The presence of VacA in the ER and its trafficking to the Golgi apparatus was confirmed in HeLa cells, identifying these two cellular compartments as novel VacA target structures.     

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin-associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co-transfected with CagA or HspB. Moreover, VacA over-expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor-mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co-operate to execute cell death with a prevalence of the mitochondrial pathways. Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of human gastric epithelial cells.     

Instrumental Role of Helicobacter pylori γ-Glutamyl Transpeptidase in VacA-Dependent Vacuolation in Gastric Epithelial Cells

Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.     

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.     

Methods to monitor autophagy in H. pylori vacuolating cytotoxin A (VacA)-treated cells

Helicobacter pylori is a gram negative pathogen that infects at least half of the world’s population and is associated not only with gastric cancer but also with other diseases such as gastritis and peptic ulcers. Indeed, H. pylori is considered the single most important risk factor for the development of gastric cancer. The vacuolating cytotoxin, VacA, secreted by H. pylori promotes intracellular survival of the bacterium and modulates host immune responses. In a recent study, we reported that VacA induces autophagy. Multilamellar autophagosomes are detected in gastric epithelial cells that are distinct from the large vacuoles formed by VacA. Furthermore, inhibition of autophagy stabilizes VacA and reduces vacuolation in the cells indicating that the toxin is being degraded by autophagy, thus limiting toxin-induced host cell damage. Many of the methods that were used for this study are commonly employed techniques that were adapted for H. pylori infection and VacA intoxication. In this paper, we describe the various methods and specific protocols used for the assessment and monitoring of autophagy during H. pylori infection.