Pylori CagA Protein Research Articles

Immunohistochemical Differentiation between Western and East Asian Types of CagA-Positive Helicobacter pylori in Gastric Biopsy Samples.

Cag A-positive Helicobacter pylori isolated from human gastric mucosa is categorized as a Western or East Asian allele-type based on whether the cagA gene encodes an EPIYA-C or EPIYA-D motif. We aimed to differentiate between the 2 types of H. pylori by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) gastric biopsy samples. We developed 2 monoclonal antibodies (mAbs) that detect either the EPIYA-C or EPIYA-D motif of the H. pylori CagA protein by IHC using FFPE tissues. FFPE tissue sections from 30 Japanese and 39 Brazilian gastric biopsy samples with H. pylori infection confirmed by Giemsa staining (moderate/severe in the Sydney classification system) were examined by IHC with the novel mAbs followed by polymerase chain reaction (PCR) for EPIYA-C or EPIYA-D using DNA extracted from adjacent tissue sections. Differentiation among Western and East Asian types and CagA-negative H. pylori was successful in most (97%) samples by IHC with the novel mAbs and commercially available mAbs that react with a species-specific lipopolysaccharide or a common CagA motif of H. pylori. The detection status of EPIYA-C/D motifs by IHC with the novel mAbs was consistent with the PCR results in 61 (88%) of 69 samples: EPIYA-C(+)/D(-) in zero Japanese and 26 Brazilian samples, EPIYA-C(-)/D(+) in 26 Japanese and 1 Brazilian sample, and EPIYA-C(-)/D(-) in 1 Japanese and 7 Brazilian samples. The detection sensitivity and specificity of IHC with each novel mAb compared with the PCR results were, respectively, 84% and 97% for EPIYA-C, and 97% and 95% for EPIYA-D. The novel mAbs specific to each EPIYA-C or EPIYA-D motif differentiated between Western and East Asian types of CagA-positive H. pylori by IHC using FFPE tissues. Applying these novel mAbs to large numbers of archived pathology samples will contribute to elucidating the association of these allele types with gastric cancer.

Comparative assessment of the results of detection of СagА-positive strains of Helicobacter pylori by molecular-genetic and immunochromatographic methods in different biological materials.

A comparative analysis of the detection of CagA-positive strains of H. pylori by immunochromatographic and molecular genetic methods was carried out. We used H. pylori strains isolated from individuals with diseases of the gastrointestinal tract. The immunochromatographic method was implemented using a developed experimental model of an immunochromatographic test system for detecting the H. pylori CagA protein in various biological materials. Determination of the pathogenicity gene cagA of H. pylori was carried out using the «Helikopol SA» test system («Litekh», Russia). The assessment of the comparability of the results of detecting CagA-positive strains of H. pylori was carried out using statistical methods: Monte-Carlo, calculation of the chi-square test (χ2) and Kendall's τ-b and Somer's d coefficients. Statistical analysis was performed using the software packages «Microsoft Office Excel», «Statistica 10.0», «WinBUGS 1.4.0.» The study showed the absence of a statistically significant difference and the presence of a direct strong correlation between the results of detecting CagA-positive strains by molecular genetic and immunochromatographic methods, which indicates that these methods provide similar results in identifying highly pathogenic strains of H. pylori.

A principal component regression model for predicting phytochemical binding to the H. pylori CagA protein

Helicobater pylori is an important causative factor in the pathogenesis of multiple gastrointestinal diseases. One of the factors responsible for the virulence of H. pylori is the cagA protein, which can interfere with a number of cellular signaling processes once this protein is transferred inside the host cell. Thus, inhibiting the interaction of the cagA protein with the host cell membrane using small molecular inhibitors appears to be a promising pharmacological strategy. In this study, a predictive model for the binding free energy of natural compounds towards the cagA protein is presented. The formulated model which is built on principal component—multiple linear regression demonstrates reliable accuracy (r2test = 0.92, RMSEtest = 0.483), while only requiring five independent variables for the prediction. It was further noted that topological descriptors had the greatest influence on the generated principal components which served as the predictors. The created regression model can help promote and accelerate the discovery of natural compounds as cagA binders for the development of anti-H. pylori agents.

Changes in Seroprevalence of Helicobacter pylori Infection over 20 Years in Jinju, Korea, from Newborns to the Elderly.

BackgroundThe objective of this study was to examine changes in the prevalence of cytotoxic-associated gene A (CagA) positive Helicobacter pylori infection in Jinju, Korea, over the last 20 years.MethodsThree cross-sectional analyses were conducted concurrently. A total of 1,305 serum samples were collected from 1994–1995, 2004–2005, and 2014–2015, respectively. The presence of immunoglobulin (Ig) G, IgA, and IgM antibodies against H. pylori CagA protein was examined by western blotting.ResultsOverall, seropositivity for anti-CagA IgG antibody was significantly decreased from 63.2% to 42.5% over the last 20 years (P < 0.001). Anti-CagA IgG seropositivities in children and young adults aged 10–29 years decreased from 1994 (60.0%–85.0%) to 2015 (12.5%–28.9%). The age when plateau of increasing IgG seropositivity was reached in each study period shifted from the 15–19 year-old group in 1994–1995 (85.0%) to the 40–49 year-old group in 2014–2015 (82.5%). Overall seropositive rates of anti-CagA IgA and IgM antibodies did not change significantly either over the last 20 years.ConclusionH. pylori infection rate in children and young adults declined over 20 years in Jinju, probably due to improved sanitation, housing, or economy.

English

cagA gene, a marker for the cag pathogenicity island (CagPAI) and a virulent factor in Helicobacter pylori infection codes for 120 to 145 kDa protein that is associated with cytotoxin production and more severe clinical outcomes. The aim of this study was to determine whether any correlation exists between H. pylori CagA protein and the endoscopic findings among dyspeptic patients from South-western, Nigeria and also to investigate the evolutionary relationships among H. pylori cagA gene with the GenBank strains. A total of one hundred and twenty four H. pylori positive isolates were amplified for the detection of cagA by polymerase chain reaction (PCR) and H. pylori isolates positive for cagA were further evaluated for protein expression using western blotting analysis. Also, DNA sequencing, blasting and phylogenetic analysis were performed on twelve selected isolates positive for cagA. cagA gene was detected in all the 124 samples (100%) and protein expressions of cagA by western blotting analysis was 88.7% (110/124). There was a high prevalence (91.7%) of expressed cagA strains in patients with positive endoscopic lesions than those with normal endoscopic findings (85.7%); however, association was not statistically significant (P&gt;0.05). Also, the expressed cagA had no statistically significant association with all the positive endoscopic findings (P&gt;0.05). Phylogenetic analysis of the selected H. pylori cagA gene showed high similarity with some GenBank strains of western cagA H. pylori. This study shows that CagA is high among dyspeptic patients in Nigeria but with no statistically significant association with the endoscopic findings. Key words: Protein expression, CagA, protein, GenBank, dyspeptic.&nbsp

3241 Expression of the Helicobater pylori CagA protein is closely associated with Helicobater pylori-dependence of early-stage high-grade gastric lymphoma

3241 Expression of the Helicobater pylori CagA protein is closely associated with Helicobater pylori-dependence of early-stage high-grade gastric lymphoma

Repressed TGF-β signaling through CagA-Smad3 interaction as pathogenic mechanisms of &lt;i&gt;Helicobacter pylori&lt;/i&gt;-associated gastritis

Helicobacter pylori (H. pylori) infection causes chronic gastric inflammation, peptic ulceration, and gastric carcinogenesis, in which H. pylori cytotoxin-associated gene A (CagA) plays major pathogenic action. Since transforming growth factor-β (TGF-β) and its signaling also are principally implicated in either modulating gastric mucosal inflammatory responses or causing carcinogenesis and are attenuated after H. pylori infection, we hypothesized that dysregulated Smad signaling and repressed TGF-β might be core pathogenic mechanism for H. pylori-associated gastritis or carcinogenesis. Until now, no precise underlying mechanism how deranged TGF-β signaling developed after H. pylori infection relevant to various clinical manifestations remains unclear. In this study, we examined the molecular mechanism about the inhibition of TGF-β signaling by H. pylori CagA protein. H. pylori CagA significantly suppressed TGF-β/Smad transcriptional responses through critical inhibition of Smad3, though CagA interacted constitutively with Smad2, Smad3, and Smad4. CagA inhibited TGF-β-induced suppression of proinflammatory chemokines, such as IL-8, CXCL1 and CXCL3, as well as TGF-β-induced transcription of target genes. In conclusion, repressed TGF-β signaling associated with CagA-positive H. pylori infection could be an important determinant for the outcome of H. pylori infection. Therefore, TGF-β signaling is one of the important determinants to avoid from H. pylori CagA pathogenicity.

Computational approaches for evaluating the effect of sequence variations and the intrinsically disordered C-terminal region of the Helicobacter pylori CagA protein on the interaction with tyrosine kinase Src.

The Helicobacter pylori CagA protein was the first bacterial oncoprotein to be identified as important in the development of human malignancies such as gastric cancer. It is not clear how it is able to deregulate a set of cell control mechanisms to induce carcinogenesis following translocation into human gastric epithelial cells. It is likely, however, that structural variations in the CagA sequence alter its affinity with the host proteins inducing differences in the pathogenicity of different H. pylori strains. Using the recently elucidated N-terminal 3D structure of H. pylori CagA, information on the full cagA gene sequence, and intrinsically disordered protein structure predictions methods we evaluated the interaction of different CagA variants with the kinase Src. An automated docking followed by molecular dynamics simulations were performed to explore CagA interaction modes with Src, one of its cellular partners. The computational approach let us establish that even in the presence of the same number and type of EPIYA motifs, CagA protein can reveal different spatial distributions. Based on the lowest affinity energy and higher number of interactions it was established that the principal forces governing the CagA-Src interaction are electrostatic. Results showed that EPIYA-D models presents higher affinity with some host proteins than EPIYA-C. Thus, we highlight the importance and advantage of the use of computational tools in combining chemical and biological data with bioinformatics for modeling and prediction purposes in some cases where experimental techniques present limitations.

Abstract 1962: Helicobacter pylori cytotoxin associated protein CagA represses TGF-β signaling through interaction with Smad proteins

Abstract Helicobacter pylori infection causes chronic gastric inflammation and peptic ulceration, and is the strongest risk factor for the development of gastric cancer. The cagA (cytotoxin-associated gene A) gene which is one of the most important virulence factors of H. pylori, is believed to contribute to the gastric tissue injury and cancer development. Transforming growth factor-β (TGF-β) signaling plays a pivotal role in modulating gastric mucosal inflammatory responses. Recently, H. pylori infection has been associated with attenuated TGF-β signaling in gastric mucosa. However, the precise underlying mechanism of H. pylori infection on TGF-β signaling and inflammatory response remains unclear. In this study, we examined the mechanism of inhibition of TGF-β signaling by H. pylori CagA protein. CagA suppressed TGF-β/Smad transcriptional responses. CagA interacted constitutively with Smad proteins, and most strongly with Smad3. Expression of CagA on AGS cells inhibited TGF-β-induced down-regulation of proinflammatory chemokines, such as CXCL1, CXCL3 and IL-8, as well as TGF-β-induced transcription of target genes. These results suggest that the attenuated TGF-β signaling associated with cagA-positive H. pylori infection could be an important determinant for the degree of inflammation provoked by H. pylori. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2011-1962

S1643 The Higher Number of EPIYA-C Segments of the H. pylori cagA Protein is a Risk Factor for Gastric Cancer and the Eastern cagA-Multimerization (CM) Sequence is Associated With Tumor Diffuse Type

S1643 The Higher Number of EPIYA-C Segments of the H. pylori cagA Protein is a Risk Factor for Gastric Cancer and the Eastern cagA-Multimerization (CM) Sequence is Associated With Tumor Diffuse Type

S1644 Helicobacter pylori Infection Displays a Mixed Inflammatory/Regulatory Cytokine Profile in Children Characterized by Increased Gastric TH1, TH2, TH17 and Treg Cytokines

H. pylori CagA protein with higher number of EPIYA-C segments or EPIYA-D motif has been associated with precancerous lesions and gastric cancer, which needs to be confirmed in different regions. Recently, it has also been shown that CagA contains polymorphic CM located downstream the EPIYA-C or D segment that mediates interaction of CagA with PAR1b/MARK2 and inhibits PAR1b kinase activity disrupting tight junctions and epithelial cell polarity. Because CagA+ status is associated with increased IL-1β, IL-8 and IL-12 release, studies attempting to demonstrate In Vitro associations between IL-8 levels and the number of EPIYA-C segments have been done, but the results are discordant. Here, we evaluated whether the number of EPIYA-C segments is associated with the risk of H. pylori-related diseases adjusting for gender and age. We also evaluate the type and number of CM sequences in respect to the risk of diseases and gastric cytokine levels. We studied 521 patients with gastritis (G, n=250, 114 cagA-), duodenal ulcer (DU, n=149, 19 cagA-) and gastric carcinoma (GC, n=125, all cagA+). The number of EPIYA-C segments and CM was determined by PCR followed by sequencing. The gastric levels of IL-1β, IL-8 and IL-12 were evaluated by ELISA (Biosource, Camarillo). Patients with GC (41%) were more often infected with strains with more than one EPIYA-C segment than the patients with G (18.5%) and DU (17.0%). In the logistic analysis, the number of EPIYA C segments (OR=3.05, 95%CI=1.64-5.66, p .23), DU (p>.29) and GC (p>.20). Neither the number nor the Eastern CM pattern was associated with the diseases or cytokine levels, but both associated with diffuse (90.1%, p=.01), but not intestinal (70.1%) tumor type. Infection with strains with more than one EPIYA-C segment was significantly associated with corpus intestinal metaplasia (p<.000) and atrophy (p=.02). In conclusion the number of EPIYA-C segments but not CM is associated with increased risk of GC. Although CagA enhances IL-8 and IL-12p70 gastric levels, the number of EPIYA-C segments and CM does not affect the cytokine production. Of note, increased Eastern CM that is associated with junctional cell defects was associated with diffuse type tumor. CNPq/FAPEMIG/INCT.

Helicobacter pylori cytotoxin associated protein CagA up-regulating gastrin gene expression

Objective To study whether Helicobacter pylori CagA protein can control gastrin gene expression and the detailed mechanism. Methods First, pcDNA3. 1ZEO (-)/cagA7 was transfected into gastric cancer cell lines AGS and SGC-7901 cells. At the same time, culturing the Helicobacter pylori NCTC11637 and infecting AGS and SGC-7901 cells with it. Next, in the infected and transfecled AGS and SGC-7901 cells, respectively adding the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126 to inhibit the two signaling pathway. Untreated gastric cancer cells and empty vector transfected cells as the control. Using real-time fluorescence quantitative PCR to detect the levels of gastrin mRNA in transfected and infected cells. Results After AGS and SGC-7901 cells were transfected with pcDNA3. lZE0(-)/cagA7 and infected with NCTC11637, the results showed that the expression of gastrin mRNA increased significantly (P ＜ 0. 05) in transfected and infected cells as compared with the control group, but after adding the inhibitor AG490 and U0126 respectively, the expression of gastrin mRNA decreased significantly(P＜0.05). Conclution These results suggest that CagA may up-regulate the expression of the gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in controlling of gastrin gene expression by CagA. Key words: Helicobacter pylon; Gastrin; CagA; ERK/MAPK; JAK/STAT

Use of a novel coinfection system reveals a role for Rac1, H-Ras, and CrkII phosphorylation inHelicobacter pylori-induced host cell actin cytoskeletal rearrangements

The Helicobacter pylori CagA protein induces profound morphological changes in the host cytoskeleton and cell scattering, but the signalling involved is poorly understood. Pseudomonas aeruginosa also affects host actin cytoskeleton in a variety of ways by injecting the ExoS and ExoT toxins which encode N-terminal GTPase activating protein and C-terminal ADP-ribosyltransferase (ADPRT) activities. In this study we developed a novel coinfection assay to gain new insights into CagA effector protein functions. We found that P. aeruginosa injecting either ExoT or ExoS efficiently prevented the H. pylori-induced scattering phenotype. Both the Rho-GAP and the ADPRT domains of ExoS were needed to block the H. pylori-induced actin cytoskeletal rearrangements, whereas either domain of ExoT was sufficient for this activity. This strategy revealed common pathways subverted by different pathogens, and aided in the definition of signalling cascades that control the CagA-mediated cell scattering and elongation. We identified Crk adapter proteins, Rac1 and H-Ras, but not RhoA or Cdc42, which are the ExoS and/or ExoT targets, as crucial components of the CagA-induced phenotype. In addition, we show that ADP-ribosylation of CrkII by ExoT blocks phosphorylation of CrkII at Y-221, which is also important for the CagA-induced signalling.

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin.

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.

MPV003 Characterization of a translocation signal on the Helicobacter pylori CagA protein

MPV003 Characterization of a translocation signal on the Helicobacter pylori CagA protein

Helicobacter pylori CagA protein modulates proliferation of RGM1 rat gastric mucosal cells

Helicobacter pylori CagA protein modulates proliferation of RGM1 rat gastric mucosal cells

Serum positive cagA in patients with non-ulcer dyspepsia and peptic ulcer disease from two centers in different regions of Turkey.

To investigate and compare frequencies of serum positive cagA in patients from two separate regions of Turkey who were grouped according to the presence of peptic ulcer disease or non-ulcer dyspepsia. One hundred and eighty Helicobacter pylori-positive patients with peptic ulcer disease or non-ulcer dyspepsia were included in the study. One hundred and fourteen patients had non-ulcer dyspepsia and 66 had peptic ulcer disease (32 with gastric ulcers and/or erosions and 34 with duodenal ulcers). Each patient was tested for serum antibody to H. pylori cagA protein by enzyme immunoassay. The total frequency of serum positive cagA in the study group was 97.2 %. The rates in the patients with peptic ulcers and in those with non-ulcer dyspepsia were 100 % and 95.6 %, respectively. These results were similar to those reported in Asian studies, but higher than those that have been noted in other studies from Turkey and Western countries. The high rates of serum positive cagA in these patients with peptic ulcer disease and non-ulcer dyspepsia were similar to results reported in Asia. The fact that there was high seroum prevalence regardless of ulcer status suggests that factors other than cagA might be responsible for ulceration or other types of severe pathology in H. pylori-positive individuals.

How H. pylori Leads Cells Astray

The gastric bacterium Helicobacter pylori is associated with gastritis, gastroduodenal ulcers, gastric adenocarcinoma, and gastric lymphoma. The cytotoxin-associated gene A antigen (CagA) is translocated into gastric epithelial cells, where it undergoes tyrosine phosphorylation and triggers cell scattering and morphological changes, and it has been implicated in H. pylori 's pathogenic effects. The mechanism by which CagA affects cell behavior and morphology, however, remainsunclear. Mimuro et al. determined that CagA interacted with the adaptor protein Grb2 (growth factor receptor-binding protein 2) in AGS gastric adenocarcinoma cells independently of having undergone tyrosine phosphorylation, which leads to activation of the mitogen-associated protein kinase pathway and to cell scattering and proliferation. Using CagA mutants expressed in H. pylori that lacked endogenous CagA, the authors determined that substituting phenylalanine for tyrosine in the phosphorylated region of CagA (PY region) did not interfere with CagA-evoked scattering in infected cells, whereas deletion of the PY region abolished cell scattering. Western analysis using fusion proteins containing Grb2 domains indicated that both phosphorylated and nonphosphorylated CagA bound to Grb2, as did the phenylalanine-substituted CagA mutant, but not the mutant lacking the PY region. A dominant-negative Grb2 mutant that interfered with Ras activation by Grb2 inhibited H. pylori -induced cell scattering. Consistent with Ras pathway implication in the response, a sustained late phase of mitogen-activated and extracellular signal-regulated kinase kinase (MEK) activation in response to H. pylori infection depended on CagA expression. Analysis of the effects of various CagA constructs stably transfected into MDCK cells implicated the PY region in CagA-induced MEK phosphorylation, as well as scattering and proliferation. H. Mimuro, T. Suzuki, J. Tanaka, M. Asahi, R. Haas, C. Sasakawa, Grb2 is a key mediator of Helicobacter pylori CagA protein activities. Mol. Cell 10 , 745-755 (2002). [Online Journal]

Cells Stabbed in the Gut

Helicobacter pylori is a prevalent human parasite (infecting half the world's population) and is linked to a variety of gut disorders, including severe gastritis and gastric carcinoma. Higashi et al. have elucidated the steps taken by the bacterial CagA protein that transform host cells. After CagA protein is injected by H. pylori into host cells, it is phosphorylated on tyrosine residues by host kinases. Phosphorylated CagA then binds to the Src homology 2 (SH2) domain-containing tyrosine phosphatase (SHP-2), which stimulates SHP-2 translocation to the cell surface where it displays its phosphatase activity. Active membrane-associated SHP-2 then stimulates morphological changes in the host cell that are the prelude to cellular transformation. H. Higashi, R. Tsutsumi, S. Muto, T. Sugiyama, T. Azuma, M. Asaka, M. Hatakeyama, SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 295 , 683-686 (2002). [Abstract] [Full Text]

Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti-phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).