Pylori CagA Research Articles

Su1829 BIoinformatic Approach to Mapping Global H. pylori cagA Sequences for Targeted Vaccine Development

Su1829 BIoinformatic Approach to Mapping Global H. pylori cagA Sequences for Targeted Vaccine Development

695 H. pylori cagY Rearrangements Modify Function of the cag Type 4 Secretion System in Response to Oncogenic Changes in the Gastric Microenvironment

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

Helicobacter pylori: correlation of the virulence marker iceA allele with clinical outcome in a high prevalence area

ABSTRACTThe association of Helicobacter pylori virulence marker ‘induced by contact with epithelium A’ (iceA) allele types was determined in H.pylori-related diseases and virulence markers. Gastric biopsies were obtained at EGD from patients for culture, histopathology and polymerase chain reaction (PCR) for iceA types, cagA and vacA alleles. Two hundred and eighty-four H. pylori isolates were examined. iceA type1 was positive in 177 (62%) and iceA type 2 in 158 (56%). In iceA type 2, gastric ulcer was present in 34 (21%) (P<0.001) and carcinoma in 28 (25%) (P=0.002), compared to nine (8%) and 2 (2%) in iceA type 2-negative cases. For iceA type 2, 139 (88%) were associated with chronic active gastritis compared to 95 (75%) (P=0.006) in iceA type 2- negative. H. pylori cagA was positive in 101 (64%) iceA type 2 strains compared to 57 (45%) in negative strains (P=0.002). H. pylori iceA type 2 was dominant and associated with cagA, chronic active inflammation, gastric ulcer and carcinoma.

Innate immunity components and cytokines in gastric mucosa in children with Helicobacter pylori infection.

Purpose. To investigate the expression of innate immunity components and cytokines in the gastric mucosa among H. pylori infected and uninfected children. Materials and Methods. Biopsies of the antral gastric mucosa from children with dyspeptic symptoms were evaluated. Gene expressions of innate immunity receptors and cytokines were measured by quantitative real-time PCR. The protein expression of selected molecules was tested by immunohistochemistry. Results. H. pylori infection did not lead to a significant upregulation of MyD88, TLR2, TLR4, CD14, TREM1, and TREM2 mRNA expression but instead resulted in high mRNA expression of IL-6, IL-10, IFN-γ, TNF-α, and CD163. H. pylori cagA(+) infection was associated with higher IL-6 and IL-10 mRNA expression, as compared to cagA(−) strains. H. pylori infected children showed increased IFN-γ and TNF-α protein levels. IFN-γ mRNA expression correlated with both H. pylori density of colonization and lymphocytic infiltration in the gastric mucosa, whereas TNF-α protein expression correlated with bacterial density. Conclusion. H. pylori infection in children was characterized by (a) Th1 expression profile, (b) lack of mRNA overexpression of natural immunity receptors, and (c) strong anti-inflammatory activities in the gastric mucosa, possibly resulting from increased activity of anti-inflammatory M2 macrophages. This may explain the mildly inflammatory gastric inflammation often observed among H. pylori infected children.

Prevalence of Helicobacter pylori cagA genotype among dyspeptic patients in Southern Thailand

Prevalence of Helicobacter pylori cagA genotype among dyspeptic patients in Southern Thailand

Prevalence of Helicobacter pylori cagA genotype among dyspeptic patients in Southern Thailand

Abstract Objective To investigate the prevalence of Helicobacter pylori ( H. pylori ) infection in dyspepsia patients and its relation to virulence factor cagA gene. Methods In total, 110 gastric biopsies from dyspeptic patients were comparatively studied using rapid urease test and multiplex PCR. Multiplex PCR detected three genes of 16S rRNA, cagA, and ureC. Results H. pylori was detected in 14 gastric biopsies (13%). Significantly higher number of female were infected. Furthermore, cagA gene was found in all H. pylori –positive specimens. In addition, the result indicated that the multiplex PCR with annealing temperature at 57 oC was able to effectively amplify specific products. Conclusion The results confirmed that high preva¬lence of cagA gene in H. pylori among dyspeptic patients in Southern Thailand.

Abstract 3180: Gastrokine 1 inhibits Helicobacter pylori CagA-induced gastric carcinogenesis

Abstract Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell mortality, and stimulates gastric carcinogenesis. We investigated the effects of CagA and GKN1 on cell proliferation, apoptosis, ROS production, epithelial-mesenchymal transition (EMT) and cell migration in CagA or GKN1 transfected gastric epithelial cells and tissues from humans and mice infected with H. pylori. We studied whether H. pylori CagA regulates gene copy number and expression of GKN1 in gastric cells and tissues of both humans and mice. On a molecular level, H. pylori CagA induced increased cell proliferation, ROS production, anti-apoptotic activity, and cell migration and invasion, and activation of NF-κB and PI3K/Akt signaling pathways and epithelial mesenchymal transition-related proteins. In addition, H. pylori CagA reduced GKN1 gene copy number and expression in gastric cells and tissues of humans and mice. However, GKN1 overexpression successfully suppressed these carcinogenic effects through directly binding to CagA. These study identifies GKN1 as pharmacological target that inhibits the development and progression of gastric cancers via suppression of H. pylori CagA-induced carcinogenic potential. Additionally, we propose that GKN1 is a potential diagnostic biomarker for H. pylori CagA status, gastritis and gastric cancers. Citation Format: Junghwan Yoon, WonSang Park, Won Seok Choi. Gastrokine 1 inhibits Helicobacter pylori CagA-induced gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2014-3180

Association between Helicobacter pylori genotypes and severity of chronic gastritis, peptic ulcer disease and gastric mucosal interleukin-8 levels: Evidence from a study in the Middle East.

BackgroundThe varied clinical presentations of Helicobacter pylori (H. pylori) infection are most likely due to differences in the virulence of individual strains, which determines its ability to induce production of interleukin-8 (IL-8) in the gastric mucosa. The aim of this study was to examine association between cagA, vacA-s1 and vacA-s2 genotypes of H. pylori and severity of chronic gastritis and presence of peptic ulcer disease (PUD), and to correlate these with IL-8 levels in the gastric mucosa.MethodsGastric mucosal biopsies were obtained from patients during esophagogastroduodenoscopy. The severity of chronic gastritis was documented using the updated Sydney system. H. pylori cagA and vacA genotypes were detected by PCR. The IL-8 levels in the gastric mucosa were measured by ELISA.ResultsH. pylori cagA and/or vacA genotypes were detected in 99 patients (mean age 38.4±12.9; 72 males), of whom 52.5% were positive for cagA, 44.4% for vacA-s1 and 39.4% for vacA-s2; and 70.7% patients had PUD. The severity of inflammation in gastric mucosa was increased with vacA-s1 (p=0.017) and decreased with vacA-s2 (p=0.025), while cagA had no association. The degree of neutrophil activity was not associated with either cagA or vacA-s1, while vacA-s2 was significantly associated with decreased neutrophil activity (p=0.027). PUD was significantly increased in patients with cagA (p=0.002) and vacA-s1 (p=0.031), and decreased in those with vacA-s2 (p=0.011). The level of IL-8 was significantly increased in patients with cagA (p=0.011) and vacA-s1 (p=0.024), and lower with vacA-s2 (p=0.004). Higher levels of IL-8 were also found in patients with a more severe chronic inflammation (p=0.001), neutrophil activity (p=0.007) and those with PUD (p=0.001).ConclusionsPresence of vacA-s1 genotype of H. pylori is associated with more severe chronic inflammation and higher levels of IL-8 in the gastric mucosa, as well as higher frequency of PUD. Patients with vacA-s2 have less severe gastritis, lower levels of IL-8, and lower rates of PUD. The presence of cagA genotype is not associated with the severity of gastritis or IL-8 induction in the gastric mucosa. The association of cagA with PUD may be a reflection of its presence with vacA-s1 genotype.

A study on the development and applications of polyclonal antibody for the detection of Helicobacter pylori in serum and saliva

In Vietnam, it’s annually estimated that there are 15,000-20,000 cases of gastric cancer, in which Helicobacter pylori (H. pylori) is the most interested agents and has been classified as a group I of carcinogens by the International Agency for Research on Cancer (IARC) based on two main toxins CagA and VacA. CagA (cytotoxin-associated gene A) encoded by the cagA gene is a protein that makes highly immune response and presents in 50-70% of H. pylori strains with a very high rate of gastric cancer. Rapid detection of H. pylori carrying cagA will contribute to warning and orienting for treatment. The aim of the study, therefore, is to produce and apply polyclonal antibodies for rapid detection of H. pylori cagA. Protein CagA was used as antigen inducing the immune response in white female mice (Swiss) at 6-8 weeks old. Indirect Elisa and Ouchterlony double immunodiffusion were performed to check antibody responses. The limit of detection and antibody titers were calculated by two fold dilution of antigen and antibody. Indirect Elisa was also used for detection of H. pylori carrying cagA in saliva and serum samples. The suitable immunogen dose was 20 mg protein/mouse. The titer of antibody was 51,200, and antibody affinity was high. Using antibody to detect H. pylori carrying cagA gene in saliva and serum samples got satisfying results.

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.

Relationship between Helicobacter pylori infection and vitiligo: a prospective study.

Helicobacter pylori is a worldwide bacteria that may affect several extra-gastric systems, including the endocrine, hematologic, vascular, respiratory, immune, and skin. Several skin diseases, including chronic urticaria, alopecia areata, psoriasis, and systemic lupus erythematosis have been found to be associated with H. pylori infection. To our knowledge, there are no data showing an association between H. pylori and vitiligo. Therefore, in this study, we wanted to evaluate the relationship between H. pylori and vitiligo. This study is a prospective study carried out in our Gastroenterology and Dermatology and Venereology departments of the Ankara Education and Research Hospital (Ankara, Turkey) between July 2013 and December 2013. Seventy-nine consecutive patients with vitiligo and 72 patients with telogen effluvium (TE) were recruited from the dermatology outpatient clinic. A total of 133 patients with vitiligo (n=68) and TE (n=65) [excluding 18 patients who had suspicious urea breath test (UBT) results] were included in the study. All individuals were tested for H. pylori IgG and CagA. Also, a UBT was performed to detect the presence of H. pylori infection. There were significantly higher rates of H. pylori positivity, H. pylori CagA, and IgG in serum in the vitiligo group than in the TE group (p<0.05). The number of patients with dyspepsia was significantly higher in the vitiligo group than in the TE group. No statistically significant relationship was seen between H. pylori positivity, CagA, H. pylori IgG, dyspepsia, and the Vitiligo Disease Activity score (p>0.05). Also, when patients with vitiligo were divided into localized and generalized types of vitiligo, there was no association between vitiligo involvement pattern and H. pylori positivity, CagA, H. pylori IgG, and dyspepsia (p>0.05). Additional studies are necessary to evaluate the effect of H. pylori eradication on the clinical course of vitiligo. Further studies are also needed to explain the relationship between H. pylori and the pathogenesis of vitiligo.

Cytotoxicity–Associated gene (CagA) Producing Helicobacter pylori Increased Risk of Developing Colorectal Carcinoma in Iraqi Patients.

Infection with Helicobacter pylori, particularly with strains positive for cytotoxicity–associated gene (CagA) gene, increases the risk of gastric adenocarcinoma and it may beassociated with carcinogenesis in extra gastric target organ. The aim of this study to explorethe possible association between CagA positive H. pylori and colorectal carcinoma anddetermine the association with clinico-pathological features.Paraffin embedded tumor specimens from (49) patients with colorectal adenocarcinoma and (30) healthy control were assessed by in situ hybridization for the expression of H. pylori CagA mRNA.Statistical analysis of H. pylori CagA positive expression revealed highly significant difference in colorectal carcinoma patients than in control group. There was no relationship between H. pylori CagA positive expression and range of clinicopathological features.Among patients infected with H. pylori CagA positive is associated with increased risk for colonic cancer.

Bioinformatics Analysis on Functional Site and Fingerprint of CagI Protein in &lt;i&gt;Helicobacter pylori&lt;/i&gt; Cag Pathogenicity Island

[Objective] This paper aims to make a bioinformatics analysis on the functional site and fingerprint of CagI protein in helicobacter pylori cag pathogenicity island and further explore the functions of CagM protein. [Methods] Use PROSITE SCAN: http://www.expasy.ch/cgi-bin/ prosite/PSScan.cgi server to search the fingerprint sequence of CagI protein and analyze and discuss the possible functions. [Results] The search of PROSITE SCAN found that CagI protein has four kinds of functional sites: N-glycosylation site, protein kinase C phosphorylation site, casein kinase II phosphorylation site, and myristoylation acting site; the search of fingerPRINTScan and printsBLAST server found sixteen fingerprint tags of CagI protein: helicobacter pylori vacuolating toxin tag; flagellin tag; ATP synthetase δ subunit tag; helicobacter pylori outer membrane protein family tag, etc. It is predicted preliminarily that CagI protein also has these functions. [Conclusion] As a kind of secretory protein, CagI protein will be secreted to the outer membrane to form the membrane protein, which will become one part of elicobacter pylori IV type production secretion pilus involved in signal transduction and material transportation and acting as a messenger or carrier, and it many have hydrolase activity and ATP/GTP enzyme activity.*Fund program: National Nature Science Foundation Project (30870096); National Science Foundation Project in Jiangsu Colleges (08KJB310001)

Bioinformatics Analysis on Subcellular Localization and Functional Site of CagM Protein in &lt;i&gt;Helicobacter pylori&lt;/i&gt; Cag Pathogenicity Island

[Objective]This paper aims to make a bioinformatics analysis on the subcellular localization and functional site of CagM protein in helicobacter pylori cag pathogenicity island and further explore the functions of CagM protein. [Methods]Apply SubLoc v1.0 program to make the subcellular localization of this protein, and use TargetP program and PSORTb version 2.0.4 to make the verification and analysis; use ANTHEPROT 5.0, Gene3D Scan Sequence software and InterPro Scan software to analyze the functional site of CagM protein, and use DOG1.0 software for the drawing. [Results]CagM is a kind of secretory protein located in the periplasmic space rather than a membrane protein; CagM has many functional sites such as N-glycosylation site, protein kinase C phosphorylation site, tyrosine kinase phosphorylation site, and myristoylation acting site as well as many PDZ III binding sites. [Conclusion]As a kind of secretory protein located in the periplasmic space, CagM protein will become the substrate of many kinds of kinases after the translation and a series of modification, and after it is activated, it will participate in the signal transmission function and the assembly of IV type secretion system, which may play a role in the collection and assembly process of other proteins.

179 Helicobacter pylori cag Secretion System-Mediated Up-Regulation of the Innate Immune Receptor TLR9

179 Helicobacter pylori cag Secretion System-Mediated Up-Regulation of the Innate Immune Receptor TLR9

Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran.

CagA is a major virulence factor of Helicobacter pylori involved in host cell modulation. The C-terminal part of CagA containing the EPIYA motifs is highly variable and is important for the biological activity of the protein. The aim of this study was consideration of the changes in cagA tyrosine phosphorylation motifs (TPMs) of H. pylori. A set of 302 H. pylori DNA samples from the Iranian population from 2006 to 2011 was selected for the proposed study. The cagA gene and its TPMs were assessed by using polymerase chain reaction (PCR) and specific primers. The prevalence of the cagA gene in our study ranged from 91.43% to 97.06% (with an average of 95.03%). Out of the cagA-positive samples, the prevalence of TPMs A and B increased from 12.5% and 23.44% to 71.2% and 63.63%, respectively. Also, the prevalence of samples infected with Western and East Asian types of H. pylori ranged from 64.06% to 5.73% for the Western type and 17.19% to 51.59% for the East Asian type. Overall, our results showed a high prevalence of the cagA gene. Also, it seems that cagA TPMs of H. pylori is undergoing a change from the Western type to the East Asian type in Iran.

The effects of H. pylori CagA on DNA amplification and protein expression status of the HER2 gene in gastric cancer.

67 Background: We investigated whether Cag A contributes to the DNA copy change and expression of the HER2 gene mRNA transcript and, consequently, affects HER2 protein expression to evaluate the significance of H. pylori Cag A and HER2 amplification in gastric cancer. Methods: We used the AGS gastric cancer and HFE-145 immortalized non-neoplastic gastric mucosa cell lines to investigate whether Cag A regulates DNA copy number and HER2 expression. We also confirmed the effects of Cag A on gastric cancer tissues. Immunohistochemistry with or without fluorescence in situ hybridization (FISH) was conducted to determine the level of HER2 protein expression based on the revised criteria in the HER2 test guidelines. Results: H. pylori CagA transfected AGS cells showed a significant increase in HER2 gene copy number and expression. In addition, Cag A expression was detected in 17 (56.7%) of 30 gastric cancer (GC) tissues, and eight (47%) showed HER2 DNA amplification of more than two-fold (P = 0.013). A positive correlation was observed among DNA copy number, the mRNA transcript, and HER2 gene protein expression (p &lt; 0.05). HER2 overexpression was detected in 12 (40%) of 30 gastric cancers by Hoffman’s criteria. Conclusions: These results suggest that H. pylori CagA may induce overexpression of the HER2 gene protein by increasing HER2 DNA and mRNA copy number. To overcome the weakness of the current HER2 assessment criteria, we propose to test HER2 DNA copy number changes with immunohistochemistry, instead of FISH.

Correction: A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

Correction: A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

Genetic Variation of Helicobacter pylori in the Oral Cavity and Stomach Detected Using Thymine Adenine Cloning in Children With Chronic Gastritis

To investigate the DNA sequence variations of Helicobacter pylori in the oral cavities and stomachs of children with chronic gastritis. Dental plaques and gargle on 235 patients were obtained before gastroscopy. Gastric H. pylori infection was diagnosed using rapid urease test or Giemsa staining. H. pylori 16S rDNA and CagA gene were analyzed using PCR methods. Thymine adenine cloning-based sequencing was performed in patients with CagA gene positive from oral cavities. The sequence alignments and V1 variable region were evaluated by comparison with the sequence of the standard H. pylori 26695 strain. Totally, 46 out of the 235 patients were positive for gastric H. pylori infection. Of the 46 patients, 26 cases had amplified H. pylori 16S rDNA in both oral (plaque and gargle) and gastric samples. Of the 26 cases, 12 patients (46.1%) were positive for the H. pylori CagA gene in the oral samples, which was significantly lower than that in the gastric mucosa (80.8%; P = 0.010). The homology of the complete sequence alignment ranged from 74.0% to 92.1% in the oral and gastric samples. The V1 region alleles (positions 75-99) had 7-22 polymorphisms (homology from 12.0% to 72.0%) in 11 patients between the oral and gastric samples, but had 100% homology in 1 patient. Our study found that distinct H. pylori strains exist in the oral cavities of children, but we would not support the hypothesis that H. pylori in the oral cavities is an important reservoir of gastric H. pylori infections in children.

Correction: Epstein Barr Virus and Helicobacter pylori Co-Infection Are Positively Associated with Severe Gastritis in Pediatric Patients.

[This corrects the article DOI: 10.1371/journal.pone.0062850.].