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CagA
Kinds of CagA Terms modified by CagA Selected AbstractsInteractions between Helicobacter pylori and other risk factors for peptic ulcer bleedingALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2002W. A. Stack To investigate the role of Helicobacter pylori, expressing the virulence marker CAGA (cytotoxin associated gene product A) in ulcer complications and its interaction with nonsteroidal anti-inflammatory drugs (NSAIDs) and other risk factors. Design: Case control study using conditional logistic regression analysis. Setting: University and City Hospitals, Nottingham. Subjects: 203 consecutive patients with ulcer bleeding and 203 age- and sex-matched controls. Results: Ulcer bleeding was more likely with positive H. pylori serology (odds ratio = 3.3, 95% CI: 1.7,6.6 for CagA positive, but only OR = 1.6, 95% CI: 0.7,3.7 for CagA negative serology), current smoking (OR 2.2, 95% CI: 1.04,4.7), aspirin , 300 mg daily (OR 7.7, 95% CI: 2.8,20.6), all other nonsteroidal anti-inflammatory drugs (NSAIDs: OR 10.6, 95% CI: 3.1,35.7 for , 1 defined daily dose lower and OR 22.6, 95% CI: 6.2,82.0 for higher doses) and past ulcer history (OR 5.6, 95% CI: 2.3,14.1). Aspirin , 300 mg daily was used by 25.1% of patients vs. 7.4% of controls. Smoking only enhanced risk in the presence of H. pylori, with a synergistic interaction (interaction odds ratio = 4.9, 2.4,9.9, P=0.002). Conversely, risks with non-aspirin NSAIDs were reduced in the presence of H. pylori, particularly if CagA-positive (interaction odds ratio=0.21, 0.05,0.9, P=0.03). Conclusions: CagA positive H. pylori infection is associated with an increased risk of ulcer bleeding. The risk from non-aspirin NSAIDs is even higher, but is less in H. pylori infected people. Low-dose aspirin is now commonly associated with ulcer bleeding. [source] Helicobacter pylori activates protein kinase C delta to control Raf in MAP kinase signalling: Role in AGS epithelial cell scattering and elongationCYTOSKELETON, Issue 10 2009Sabine Brandt Abstract Helicobacter pylori is a major etiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. Virulent H. pylori strains harbor a type IV secretion system (T4SS) encoded by the cag pathogenicity island. This T4SS injects the CagA protein into gastric epithelial cells leading to actin-cytoskeletal rearrangements followed by cell elongation and scattering. Here we report that PMA (4,-phorbol-12-myristate-13-acetate), a well-known cell-permeable activator of protein kinase C (PKC), induces a remarkably similar cellular phenotype as compared to infection with H. pylori. PKCs comprise a large family of serine/threonine kinases which are important for multiple physiological processes of host cells. We therefore investigated the role of individual PKC members and the signalling pathways involved in phenotypical outcome. Using isoform-specific silencing RNAs and pharmacological inhibitors we found that two isoforms, PKC-, and PKC-,, were essential for both PMA- and H. pylori -induced elongation phenotype. Furthermore, we provide evidence that PKC-, activity is profoundly stimulated during the course of infection using activation-specific antibodies against PKC phosphorylated at threonine residue 505 or serine residue 660. Infection with H. pylori wild-type and mutants showed that at least two bacterial factors activate PKC-, in a time-dependent manner, one of which is CagA. Immunofluorescence microscopy studies further demonstrated that phosphorylated PKC-, is accumulated and recruited to dynamic actin-structures at the cell membrane. Finally, we show that PKC-, specifically targets Raf kinase to stimulate the Erk1/2 kinase pathway, which is also crucial for phenotypical outcome. Thus, PKC-, is another important mediator of H. pylori -induced pathogenesis. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic toolFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2005Shiho Yamazaki Abstract The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian- cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian- cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA -positive; 26.8% (11/41) were Western- cagA positive and 53.7% (22/41) were East Asian- cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. [source] The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key HypothesisHELICOBACTER, Issue 3 2010Steffen Backert Abstract 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. [source] Distinctiveness of the cagA Genotype in Children and Adults with Peptic Symptoms in South ChinaHELICOBACTER, Issue 4 2009Juan Li Abstract Background:,Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3, region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. Materials and Methods:, One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3, region of CagA by conventional PCR and DNA sequencing. Results:,H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA -positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs , ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%) , were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). Conclusion:,cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2008Javier Torres Abstract The clinical outcome of Helicobacter pylori infection is determined by a complex scenario of interactions between the bacterium and the host. The main bacterial factors associated with colonization and pathogenicity comprise outer membrane proteins including BabA, SabA, OipA, AlpA/B, as well as the virulence factors CagA in the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA. The multitude of these proteins and allelic variation makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori -associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating the induction of membrane dynamics, actin-cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and antiapoptotic nuclear responses. An animal model using the Mongolian gerbil is a useful system to study the gastric pathology of H. pylori infection. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2007Shin Maeda Abstract The clinical outcome of Helicobacter pylori infection is determined by a complex interaction between the bacterium and the host. The main bacterial factors associated with pathogenicity comprise outer membrane proteins, including BabA, SabA, OipA, AlpA, and AlpB, the vacuolating cytotoxin VacA and the products of cagPAI. The multitude of putative virulence factors makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori -associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating cell transformation, cell proliferation, invasion, apoptosis/anti-apoptosis, and angiogenesis. An animal model using the Mongolian gerbil is a useful model for showing gastric pathology due to H. pylori infection which is similar to that in humans and can be used to evaluate virulence factors including CagA, host responses, and environmental factors such as salt intake. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2006Masanori Hatakeyama Abstract Much interest has been shown in the relationship between Helicobacter pylori infection and gastric carcinogenesis. It is becoming clearer that H. pylori strains carrying a functional cag pathogenicity island (cagPAI), which encodes the type IV secretion system (TFSS) and its effector CagA, play an important role in the development of gastric carcinoma. Furthermore, genetic polymorphism present in the cagA gene appears to influence the degree of an individual cagPAI-positive H. pylori to elicit gastric mucosal lesions, and this process is significantly affected by host genetic polymorphisms such as proinflammatory cytokine gene polymorphisms. Pathomechanism of gastric carcinogenesis associated with H. pylori includes bacteria,host interaction leading to morphologic alterations such as atrophic gastritis and gastrointestinal metaplasia mediated by COX-2 overexpression, cancer cell invasion, and neo-angiogenesis via TLR2/TLR9 system and transcription factors (e.g., NF-,B) activation. In addition, H. pylori infection triggers adhesion molecule expression and activity and produces an enhancement in oxidative stress interacting with gastric production of appetite hormone ghrelin and nonsteroidal anti-inflammatory drugs. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2004Paul Hofman ABSTRACT Research in the last year has provided new insights into the function of the the cag -associated type IV secretion system and the vacuolating toxin VacA. A quite new aspect was disclosed by the finding that Helicobacter pylori in Mongolian gerbils colonizes a very distinct topology in the gastric mucous layer, obviously providing optimal conditions for long-term survival. Further research activities focused on H. pylori ammonia and metal metabolism as well as on bacterial stress defence mechanisms. Differential expression of approximately 7% of the bacterial genome was found at low pH suggesting that H. pylori has evolved a multitude of acid-adaptive mechanisms. VacA was shown to interrupt phagosome maturation in macrophage cell lines as well as to modulate and interfere with T lymphocyte immunological functions. Gastric mucosa as well as the H. pylori -infected epithelial cell line AGS strongly express IL-8 receptor A and B, which might contribute to the augmentation of the inflammatory response. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. The chronic imbalance between apoptosis and cell proliferation is the first step of gastric carcinogenesis. In this regard, it was demonstrated that coexpression of two H. pylori proteins, CagA and HspB, in AGS cells, caused an increase in E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein. Taken together, we now have a better understanding of the role of different virulence factors of H. pylori. There is still a lot to be learned, but the promising discoveries summarized here, demonstrate that the investigation of the bacterial survival strategies will give novel insights into pathogenesis and disease development. [source] Is the Association Between Helicobacter pylori and Gastric Cancer Confined to CagA-Positive Strains?HELICOBACTER, Issue 3 2004Maria Held ABSTRACT Background., Infection with Helicobacter pylori is associated with an increased risk of gastric cancer. Several studies have indicated that the association differs with strain type. We aimed to find out if infection with strains lacking the virulence factor CagA is linked to gastric cancer risk. Materials and methods., In a hospital-based case,control study, we collected sera from 100 case patients with a newly diagnosed gastric adenocarcinoma and 96 control patients with diseases unrelated to H. pylori status. Antibodies to H. pylori were analyzed by enzyme-linked immunosorbent assay (ELISA), and antibodies to CagA were detected by immunoblot. Logistic regression was used to obtain odds ratios (ORs) as estimates of relative risk, adjusted for potential confounding. Results., Among the case patients, 81% were ELISA positive and 86% had antibodies to CagA. The corresponding numbers among the controls were 58% and 55%, respectively. ELISA positivity was associated with an increased risk of gastric adenocarcinoma compared to ELISA negativity (OR for gastric cancer regardless of site 3.9, 95% CI 1.9,8.2). The OR was 7.4 (95% CI 3.3,16.6) for CagA-positive relative to CagA-negative subjects. Among ELISA-positive subjects the presence of CagA antibodies increased the risk 3.6 times (95% CI 1.2,11.1). ELISA-positive CagA-negative infections were associated with a fourfold increased risk (OR = 4.2, 95% CI 1.0,17.0) compared to no infection (ELISA-negative and CagA-negative). Conclusions., Although patients with antibodies to CagA have the greatest risk of developing gastric cancer, those with CagA-negative infections run a significantly greater risk than uninfected persons. [source] Serological Responses of FldA and Small-Molecular-Weight Proteins of Helicobacter pylori: Correlation with the Presence of the Gastric MALT TissueHELICOBACTER, Issue 1 2004Hsiao-Bai Yang ABSTRACT Purpose., We tested whether the serological response to Flavodoxin-A (FldA) protein and anti- Helicobacter pylori immunoblots correlated to the degree of mucosa-associated lymphoid tissue (MALT) in the stomach. Methods., Eighty H. pylori -infected patients with different degrees of MALT in the stomach were investigated with serum sampling and endoscopy on enrolment, the 2nd and the 12th months after anti- H. pylori therapy. All sera were tested for the anti-FldA protein and anti- H. pylori immunoblots, including 19.5, 26.5, 30, 35, 89, and 116 KDa (CagA). Regression of follicular gastritis was assessed by histology. Results., Patients with dense lymphoid follicles had higher prevalence rates of anti-FldA protein, 19.5, 26.5, and 30 KDa antibodies of H. pylori (p < .05). Histologic downgrade of MALT was observed in 25% (10/40) of patients in the 2nd month and in 60% (23/38) in the 12th month after H. pylori therapy. After H. pylori eradication, the patients with MALT and dense lymphoid follicles had significantly negative seroconversions of 19.5, 26.5, 30, and 35 KDa antibodies (p < .05), but not of CagA and FldA. Conclusion., Patients with gastric MALT and dense lymphoid follicles had different anti- H. pylori serological responses to those with scanty or an absence of lymphoid follicles. The negative seroconversion of the smaller-molecular-weight proteins, but not CagA and FldA, may correlate with the regression of MALT by H. pylori eradication. [source] Pathogenesis of Helicobacter pylori infectionHELICOBACTER, Issue 2002Markus Gerhard Five years after publication of the complete genome sequence of Helicobacter pylori, research interest is shifting from the descriptive association of virulence factors with clinical outcome in infected patients to the molecular mechanisms of virulence factor action. This is particularly noticeable for VacA and CagA, for both of which detailed understanding of the interaction with host signalling pathways has accumulated over the last year. The role of H. pylori Lewis antigens for clinical outcome was further substantiated. Various strategies of H. pylori to fool or evade the human immune system are described, which all lead to the dysfunction of specific compartments of the host cellular immune system. Finally, a number of animal models indicate that inflammation is a key factor for gastric carcinogenesis, which is finally supported by a large prospective study identifying corpus atrophy and intestinal metaplasia as precancerous conditions. [source] Helicobacter pylori CagA: Analysis of Sequence Diversity in Relation to Phosphorylation Motifs and Implications for the Role of CagA as a Virulence FactorHELICOBACTER, Issue 3 2001Doyle J. Evans Jr Abstract 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. [source] Disease-specific Helicobacter pylori Virulence Factors: The Unfulfilled PromiseHELICOBACTER, Issue S1 2000David Y. Graham A number of putative virulence factors for Helicobacter pylori have been identified including cagA, vacA and iceA. The criteria for a true virulence factor includes meeting the tests of biologically plausibility with the associations being both experimentally and epidemiologically consistent. Although disease-specific associations have been hypothesized/claimed, there are now sufficient data to conclusively state that none of these putative virulence factors have disease specificity. CagA has been claimed to be associated with increased mucosal IL-8 and inflammation, increased density of H. pylori in the antrum, duodenal ulcer (DU), gastric cancer, and protection against Barrett's cancer. Only the increase in IL-8/inflammation is direct and substantiated. Different H. pylori strains with functional cag pathogenicity islands do not vary in virulance as it has been shown that mucosal IL-8 levels are proportional to the number of cagA +H. pylori independent of the disease from which the H. pylori were obtained. It is now known that the density of either cagA + and cagA,H. pylori in the antrum of patients with H. pylori gastritis is the same. In contrast, the mean density of H. pylori in the antrum in DU is greater than in the antrum of patients with H. pylori gastritis. Of interest, the density of H. pylori is higher in the corpus of patients with H. pylori gastritis than those with DU, suggesting that acid secretion plays a critical role in these phenomena. The presence of a functional cag pathogenicity island increases inflammation and it is likely that any factor that results in an increase in inflammation also increases the risk of a symptomatic outcome. Nevertheless, the presence of a functional cag pathogenicity island has no predictive value for the presence, or the future development of a clinically significant outcome. The hypothesis that iceA has disease specificity has not been confirmed and there is currently no known biological or epidemiological evidence for a role for iceA as a virulence factor in H. pylori -related disease. The claim that vacA genotyping might prove clinically useful, e.g. to predict presentation such as duodenal ulcer, has been proven wrong. Analysis of the worldwide data show that vacA genotype s1 is actually a surrogate for the cag pathogenicity island. There is now evidence to suggest that virulence is a host-dependent factor. The pattern of gastritis has withstood the test of time for its relation to different H. pylori -related diseases (e.g. antral predominant gastritis with duodenal ulcer disease). The primary factors responsible for the different patterns of gastritis in response to an H. pylori infection are environmental (e.g. diet), with the H. pylori strain playing a lesser role. Future studies should work to eliminate potential bias before claiming disease associations. Controls must exclude regional or geographic associations related to the common strain circulation and not to the outcome. The authors must also control for both the presence of the factor and for the disease association. The study should be sufficiently large and employ different diseases and ethnic groups for the results to be robust. The findings in the initial sample (data derived hypothesis) should be tested in a new group (hypothesis testing), preferably from another area, before making claims. Finally, it is important to ask whether the results are actually a surrogate for another marker (e.g. vacA s1 for cagA) masquerading for a new finding. Only the cag pathogenicity island has passed the tests of biological plausibility (increased inflammation) and experimental and epidemiological consistency. [source] Systematic review and meta-analysis: importance of CagA status for successful eradication of Helicobacter pylori infectionALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2006T. SUZUKI Summary Background Some, but not all studies have provided evidence that the CagA status of Helicobacter pylori strains is a predictive factor for the outcome of eradication therapy. Aim To clarify the association between CagA status and eradication outcome. Methods We included studies reporting the numbers of successful and failed cases in H. pylori -eradication therapy according to the CagA status. Fourteen studies (1529 patients) were included of 325 articles identified in the search. The pooled risk ratio for H. pylori -eradication failure in CagA-negative relative to CagA-positive strains and the pooled risk difference in eradication success between the two groups were used as summary statistics. Meta-regression was used for examining the source of heterogeneity. Results The summary risk ratio for eradication failure in CagA-negative relative to CagA-positive was 2.0 (95% CI: 1.6,2.4, P < 0.001), corresponding with the summary risk difference for eradication success between the groups of 11% (95% CI: 3,19%, P = 0.011). Meta-regression analysis demonstrated that usage of polymerase chain reaction examination for CagA status and a high proportion of non-ulcer dyspepsia patients were factors for heterogeneity among studies. Conclusions Our meta-analysis confirmed the importance of the presence of CagA as a predictor for successful eradication of H. pylori. [source] Genotyping of the cagA gene of Helicobacter pylori on immunohistochemistry with East Asian CagA-specific antibodyPATHOLOGY INTERNATIONAL, Issue 4 2008Ryoko Kanada The cytotoxin-associated antigen A (CagA) of Helicobacter pylori prevalent in East Asian countries, where the mortality rate due to gastric cancer is high, has been reported to be structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. Based on the structural features of the EPIYA motifs located at the carboxyl terminal of the protein, CagA was subdivided into two types: East Asian CagA and Western CagA. A recent study suggested that immunohistochemistry with anti-East Asian-specific antibody (,-EAS Ab), which was specifically immunoreactive with East Asian CagA but not with Western CagA, may be useful for diagnosis of the cagA genotype. To further evaluate the value of this diagnostic method in terms of sensitivity, specificity, and accuracy, 143 gastric biopsy specimens with ,-EAS Ab were analyzed on immunohistochemistry and compared with the sequencing of the cagA gene. It was found that diagnosis of the cagA genotype of H. pylori on immunohistochemistry using the ,-EAS Ab was highly sensitive (sensitivity 93.2%) and specific (specificity 72.7%), suggesting that immunohistochemical diagnosis of the cagA genotype is useful for diagnosis of the cagA genotype. [source] A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian typeAPMIS, Issue 12 2009AIKO YASUDA Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA-positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori -infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up-regulated by adhesion to epithelial cells. [source] Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagACANCER SCIENCE, Issue 10 2008Huai-Sheng Lu Helicobacter pylori (H. pylori) cagA -positive strains are associated with gastritis, peptic ulcerations, and gastric adenocarcinoma. Upon delivery into gastric epithelial cells, the cagA -encoded CagA protein specifically binds and aberrantly activates SHP-2 oncoprotein in a manner that is dependent on CagA tyrosine phosphorylation. CagA-deregulated SHP-2 then elicits aberrant Erk activation while causing an elongated cell shape known as the hummingbird phenotype. In polarized epithelial cells, CagA also binds to PAR1b/MARK2 and inhibits the PAR1b kinase activity, thereby disrupting tight junctions and epithelial cell polarity independent of CagA tyrosine phosphorylation. We show here that the CagA-multimerization (CM) sequence that mediates interaction of CagA with PAR1b is not only essential for the CagA-triggered junctional defects but also plays an important role in induction of the hummingbird phenotype by potentiating CagA-SHP-2 complex formation. We also show that the CM sequence of CagA isolated from East Asian H. pylori (referred to as the E-CM sequence) binds PAR1b more strongly than that of CagA isolated from Western H. pylori (referred to as the W-CM sequence). Within Western CagA species, the ability to bind PAR1b is proportional to the number of W-CM sequences. Furthermore, the level of PAR1b-binding activity of CagA correlates with the magnitude of junctional defects and the degree of hummingbird phenotype induction. Our findings reveal that structural diversity in the CM sequence is an important determinant for the degree of virulence of CagA, a bacterial oncoprotein that is associated with gastric carcinogenesis. (Cancer Sci 2008; 99: 2004,2011) [source] Immunohistochemical diagnosis of the cagA -gene genotype of Helicobacter pylori with anti-East Asian CagA-specific antibodyCANCER SCIENCE, Issue 4 2007Tomohisa Uchida Cytotoxin-associated antigen A (CagA) protein produced by Helicobacter pylori is proposed to be associated with the pathogenesis of gastric cancer as well as gastritis and gastroduodenal ulcer. It has been reported that the CagA of H. pylori widespread in East Asian countries, where the mortality rate due to gastric cancer is high, is structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. In this study, we generated an antibody, East Asian CagA-specific antibody (,-EAS Ab), which is specifically immunoreactive with East Asian CagA but not with Western CagA. The CagA was immunohistochemically detected at the surface of the gastric mucosa. Interestingly, positive immunoreactivity was also detected in the nucleus and cytoplasm of the infected gastric epithelium, suggesting that CagA may play some pathogenic role in both the nucleus and cytoplasm. Immunohistochemistry of 47 gastric biopsy specimens detected East Asian CagA-positive H. pylori in 43 cases. In 46 of the 47 cases examined, the data obtained by immunohistochemistry were completely consistent with those obtained by sequencing of the cagA gene of the isolated strain, suggesting that our immunohistochemical method is reliable and useful for diagnosis of the cagA genotype. (Cancer Sci 2007; 98: 521,528) [source] Conditional gene silencing utilizing the lac repressor reveals a role of SHP-2 in cagA -positive Helicobacter pylori pathogenicityCANCER SCIENCE, Issue 5 2004Megumi Higuchi RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-,-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach. [source] Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infectionCELLULAR MICROBIOLOGY, Issue 5 2010Tamia K. Lapointe Summary Helicobacter pylori infects more than half of the human population worldwide. In the absence of treatment, this persistent infection leads to asymptomatic gastritis, which in some cases can progress into gastric ulcers and adenocarcinomas. The host,microbial interactions that govern the clinical outcome of infection remain incompletely understood. H. pylori is known to disrupt gastric epithelial tight junctions, which may represent a significant component of disease pathogenesis. The present study demonstrates that H. pylori disrupt epithelial tight junctional claudin-4 in a Rho kinase (ROCK)-dependent manner in human gastric epithelial (HGE-20) cell monolayers, independently of the virulence factors CagA and VacA, and without altering claudin-4 transcription. In the same epithelial cell model, interleukin (IL)-1,, mediated a similar ROCK-dependent pattern of tight junction disruption. Further experiments revealed that H. pylori infection induced IL-1 receptor type I (IL-1RI) phosphorylation, independently of epithelial secretion of its endogenous ligands IL-1,, IL-1, or IL-18. Finally, inhibition of IL-1RI activation prevented H. pylori -induced ROCK activation and claudin-4 disruption. Taken together, these findings identify a novel pathophysiological mechanism by which H. pylori disrupts gastric epithelial barrier structure via IL-1RI-dependent activation of ROCK, which in turn mediates tight junctional claudin-4 disruption. [source] 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 VacACELLULAR MICROBIOLOGY, Issue 3 2009Nicole Tegtmeyer Summary 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 (cagPAI) and the vacuolating cytotoxin VacA are key players in disease development. The cagPAI 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-,, 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. [source] H. pylori selectively blocks EGFR endocytosis via the non-receptor kinase c-Abl and CagACELLULAR MICROBIOLOGY, Issue 1 2009Bianca Bauer Summary Helicobacter pylori infection is a primary cause of peptic ulcers and is associated with gastric carcinogenesis. The H. pylori -induced pathophysiology may be linked to the deregulation of EGFR signalling. Elevated mucosal levels of EGF and the EGFR have been found in antral gastric biopsies of H. pylori -infected patients. A critical mechanism for regulating EGFR signalling is ligand-induced endocytosis. The internalized receptor recycles back to the plasma membrane for continued signalling or is targeted for degradation terminating receptor signalling. Here, we show that H. pylori blocks EGFR endocytosis and receptor degradation upon prolonged infection of gastric epithelial cells. Moreover, this inhibition occurs via a CagA-dependent, but CagA phosphorylation-independent activation of the non-receptor kinase c-Abl, which in turn phosphorylates the EGFR target site pY1173. This suggests a novel CagA-induced host cell response that is independent of CagA tyrosine phosphorylation. Our data indicate an intriguing strategy of H. pylori in host cell manipulations by altering selective receptor populations via a CagA-dependent endocytic mechanism. Furthermore, we identified a new role for c-Abl in phosphorylation of the EGFR target site pY1173 during H. pylori infection. [source] Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarityCELLULAR MICROBIOLOGY, Issue 3 2008Zaher Zeaiter Summary Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation. [source] Helicobacter pylori,host cell interactions mediated by type IV secretionCELLULAR MICROBIOLOGY, Issue 7 2005Kevin M. Bourzac Summary Helicobacter pylori is a human-specific gastric pathogen that colonizes over half the world's population. Infection with this bacterium is associated with a spectrum of gastric pathologies ranging from mild gastritis to peptic ulcers and gastric cancer. A strong predictor of severe disease outcome is infection with a bacterial strain harbouring the cag (cytotoxin associated gene) pathogenicity island (PAI), a 40 kb stretch of DNA that encodes homologues of several components of a type IV secretion system (TFSS). One gene within the cag PAI, cagA, has been shown to encode a substrate for the TFSS which is translocated into host cells and causes multiple changes in host cell signalling. Here we review recent advances in the characterization of type IV secretion, the activities of CagA and CagA-independent effects of the TFSS, which are contributing to our understanding of H. pylori pathogenesis. [source] Relationships between cagA, vacA, and iceA genotypes of Helicobacter pylori and DNA damage in the gastric mucosaENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2004Marcelo S.P. Ladeira Abstract Helicobacter pylori (H. pylori) is believed to predispose carriers to gastric cancer by inducing chronic inflammation. The inflammatory processes may result in the generation of reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the relationships between DNA damage in the gastric mucosa and cagA, vacA, and iceA genotypes of H. pylori. The study was conducted with biopsies from the gastric antrum and corpus of 98 H. pylori -infected and 26 uninfected control patients. H. pylori genotypes were determined by PCR and DNA damage was measured in gastric mucosal cells by the Comet assay (single cell gel electrophoresis). All patients were nonsmokers, not abusing alcohol, and not using prescription or recreational drugs. Levels of DNA damage were significantly higher (P < 0.0001) in the H. pylori -infected patients than in uninfected patients. In comparison with the level of DNA damage in the uninfected controls, the extent of DNA damage in both the antrum (OR = 8.45; 95% CI = 2.33,37.72) and the corpus (OR = 6.55; 95% CI = 2.52,17.72) was related to infection by cagA+/vacAs1m1 and iceA1 strains. The results indicate that the genotype of H. pylori is related to the amount of DNA damage in the gastric mucosa. These genotypes could serve as biomarkers for the risk of extensive DNA damage and possibly gastric cancer. Environ. Mol. Mutagen. 44:91,98, 2004. © 2004 Wiley-Liss, Inc. [source] Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic toolFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2005Shiho Yamazaki Abstract The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian- cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian- cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA -positive; 26.8% (11/41) were Western- cagA positive and 53.7% (22/41) were East Asian- cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. [source] cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groupsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Mohamed Ramelah Abstract Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3, region of cagA gene were examined among 192 Malaysian H. pylori cagA -positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621,651 bp), type B (732,735 bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p > 0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific- cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p < 0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p < 0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection. [source] Presence of High Numbers of Transcriptionally Active Helicobacter pylori in Vomitus from Bangladeshi Patients Suffering from Acute GastroenteritisHELICOBACTER, Issue 4 2009Anders Janzon Abstract Background:,Helicobacter pylori is one of the most prevalent human bacterial pathogens; however, its transmission pathways remain unknown. New infections of H. pylori during outbreaks of gastroenteritis have been suggested previously, and to explore this transmission route further H. pylori was quantified in vomitus and diarrheal stool of patients suffering from acute gastroenteritis in Dhaka, Bangladesh. Materials and Methods:, Vomitus and stool samples from 28 patients seeking care at the International Centre for Diarrhoeal Disease Research hospital were analyzed for presence of H. pylori and other pathogens using quantitative culturing, real-time polymerase chain reaction (PCR), and H. pylori stool antigen test. Bacterial gene expression was analyzed using reverse transcriptase real-time PCR. Results:, The results of real-time PCR show that 23 (88%) of the 26 vomitus samples and 17 (74%) of the 23 stool samples were H. pylori positive, while stool antigen test show that 14 (67%) of the 21 stool samples were H. pylori positive. H. pylori could not be isolated by culture. Analysis using quantitative culture and real-time PCR to detect Vibrio cholerae showed strong correlation between these methods, and validating real-time PCR. Analysis of H. pylori virulence gene transcription in vomitus, diarrheal stool, antral and duodenal biopsy specimens, and in vitro cultures showed that cagA, flaA, and ureA were highly transcribed in vomitus, biopsy specimens, and cultures, whereas hpaA and vacA were expressed at lower levels. No H. pylori gene expression was detected in diarrheal stool. Conclusions:, We conclude that high numbers of transcriptionally active H. pylori are shed in vomitus, which indicates that new infections may be disseminated through vomiting. [source] Distinctiveness of the cagA Genotype in Children and Adults with Peptic Symptoms in South ChinaHELICOBACTER, Issue 4 2009Juan Li Abstract Background:,Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3, region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. Materials and Methods:, One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3, region of CagA by conventional PCR and DNA sequencing. Results:,H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA -positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs , ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%) , were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). Conclusion:,cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China. [source] |