Dataset Information

CagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

ABSTRACT: The aim of this work was to determine current cagA gene EPIYA motifs present in Colombian Helicobacter pylori isolates using a fast and reliable molecular test. DNA from eighty-five Helicobacter pylori-cagA positive strains were analyzed. Strains were obtained from patients diagnosed with functional dyspepsia at Clínica Fundadores in Bogotá. The 3' region of the cagA gene was amplified through conventional Polymerase Chain Reaction (PCR). Obtained amplicons were sequenced using the Sanger method and analyzed with bioinformatics tools. Additionally, a significant Spearman correlation coefficient was determined between the patients' age and the number of EPIYA-C repeats; with p values < 0.05 considered significant. Estimates were obtained using a 95% CI. The 3´ variable region of the cagA gene was amplified and PCR products of the following sizes corresponded to the following EPIYA motifs: 400 bp: EPIYA AB, 500 bp: EPIYA ABC, 600 bp: EPIYA ABCC and 700 bp: ABCCC. A single PCR band was observed for 58 out of 85 Helicobacter pylori isolates, with an EPIYA distribution motif as follows: 7/85 AB (8.2%), 34/85 ABC (40%), 26/85 ABCC (30.6%) and 18/85 ABCCC (21.2%). However, in 27 out of 85 Helicobacter pylori isolates, two or more bands were observed, where the most predominant cagA genotype were ABC-ABCC (26%, 7/27) and ABCC-ABCCC (22.2%, 6/27). A direct proportionality between the number of EPIYA-C repeats and an increase in the patients' age was observed, finding a greater number of EPIYA ABCC and ABCCC repeats in the population over 50 years old. All isolates were of the Western cagA type and 51.8% of them were found to have multiple EPIYA-C repeats. These standardized molecular test allowed to identify the number of EPIYA C motifs based on band size.

SUBMITTER: Rodriguez Gomez ER

PROVIDER: S-EPMC6953880 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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cagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

Rodríguez Gómez Eliana Rocío ER Otero Regino William W Monterrey Pedro A PA Trespalacios Rangel Alba Alicia AA

PloS one 20200110 1

The aim of this work was to determine current cagA gene EPIYA motifs present in Colombian Helicobacter pylori isolates using a fast and reliable molecular test. DNA from eighty-five Helicobacter pylori-cagA positive strains were analyzed. Strains were obtained from patients diagnosed with functional dyspepsia at Clínica Fundadores in Bogotá. The 3' region of the cagA gene was amplified through conventional Polymerase Chain Reaction (PCR). Obtained amplicons were sequenced using the Sanger method ...[more]

PMID: 31923209

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Project description:BackgroundHelicobacter pylori chronic infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. Cytotoxin-associated gene A (cagA)-positive H. pylori strains increase the risk of gastric pathology. The carcinogenic potential of CagA is linked to its polymorphic EPIYA motif variants. The goals of this study were to investigate the frequency of cagA-positive Helicobacter pylori in Mexican patients with gastric pathologies and to assess the association of cagA EPIYA motif patterns with peptic ulcer and gastric cancer.MethodsA total of 499 patients were studied; of these, 402 had chronic gastritis, 77 had peptic ulcer, and 20 had gastric cancer. H. pylori DNA, cagA, and the EPIYA motifs were detected in total DNA from gastric biopsies by PCR. The type and number of EPIYA segments were determined by the electrophoretic patterns. To confirm the PCR results, 20 amplicons of the cagA 3' variable region were sequenced, and analyzed in silico, and the amino acid sequence was predicted with MEGA software, version 5. The odds ratio (OR) was calculated to determine the associations between the EPIYA motif type and gastric pathology and between the number of EPIYA-C segments and peptic ulcers and gastric cancer.ResultsH. pylori DNA was found in 287 (57.5%) of the 499 patients, and 214 (74%) of these patients were cagA-positive. The frequency of cagA-positive H. pylori was 74.6% (164/220) in chronic gastritis patients, 73.6% (39/53) in peptic ulcer patients, and 78.6% (11/14) in gastric cancer patients. The EPIYA-ABC pattern was more frequently observed in chronic gastritis patients (79.3%, 130/164), while the EPIYA-ABCC sequence was more frequently observed in peptic ulcer (64.1%, 25/39) and gastric cancer patients (54.5%, 6/11). However, the risks of peptic ulcer (OR = 7.0, 95% CI = 3.3-15.1; p < 0.001) and gastric cancer (OR = 5.9, 95% CI = 1.5-22.1) were significantly increased in individuals who harbored the EPIYA-ABCC cagA gene pattern.ConclusionscagA-positive H. pylori is highly prevalent in southern Mexico, and all CagA variants were of the western type. The cagA alleles that code for EPIYA-ABCC motif patterns are associated with peptic ulcers and gastric cancer.

Project description:The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors--in particular various SH2-domain containing human proteins--thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial anti-phosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.

Project description:BackgroundHighly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming.ResultsThis study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells.ConclusionsThe results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

Dataset Information

CagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

Publications

cagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

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