The validation of the Helicobacter pylori CagA typing by immunohistochemistry: nationwide application in Indonesia.
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ABSTRACT:
SUBMITTER: Miftahussurur M
PROVIDER: S-EPMC7486129 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (sla, slb, and s2 alleles), the vacA m region (ml and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type slb, whereas most Dutch patients (61%) contained type sla (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type sl (sla or slb; P = 0.008) and cagA (P = 0.003) genes.
| S-EPMC104813 | biostudies-literature
Project description:To develop a novel Helicobacter pylori (H. pylori) CagA antibody enzyme-linked immunosorbent assay (ELISA) suitable for detecting serum anti-CagA antibodies with high sensitivity.Recombinant East Asian-type CagA protein was purified and immobilized for ELISA. Serum samples from 217 Vietnamese individuals (110 H. pylori-infected and 107 uninfected individuals) were applied. Conventional ELISA from Western-type CagA and our East Asian-type CagA ELISA were evaluated by comparing 38 subjects with the Western-type genotype and 72 subjects with the East Asian-type cagA genotype. Histological scores of the gastric mucosa were determined using the updated Sydney System to examine the relationship with anti-CagA antibody titers.Recombinant 70-100 kDa fragments were immobilized on the ELISA plate. In ROC analysis, the area under the curve of our East Asian-type CagA ELISA was comparable to that of conventional CagA ELISA. The sensitivity of the two ELISAs differed depending on the cagA genotype. The sensitivity of East Asian-type CagA ELISA was higher for subjects infected with East Asian-type cagA H. pylori (P < 0.001), and the sensitivity of the conventional CagA ELISA tended to be higher for subjects infected with Western cagA H. pylori (P = 0.056). The titer of anti-CagA antibody tended to correlate with monocyte infiltration scores (r = 0.25, P = 0.058) and was inversely correlated with H. pylori density (r = -0.26, P = 0.043).The novel ELISA is useful to detect anti-CagA antibodies in East Asian countries, and the titer may be a marker for predicting chronic gastritis.
| S-EPMC5221286 | biostudies-literature
Project description:Approximately 50% of Helicobacter pylori strains produce a cytotoxin that is encoded by vacA and that induces vacuolation of eukaryotic cells. Mosaicism in vacA alleles was reported, and there are three different families of vacA signal sequences (s1a, s1b, and s2) and two different families of middle-region alleles (m1 and m2). In addition, the vacA genotype of a strain is associated with its cytotoxin phenotype and its capacity to induce peptic ulceration. To clarify the strain diversity of H. pylori in Japan, 87 Japanese clinical isolates of H. pylori (40 from patients with chronic atrophic gastritis, 25 from patients with duodenal ulcer, 16 from patients with gastric ulcer, 3 from patients with both duodenal and gastric ulcers, and 3 from patients with intestinal type gastric cancer) were characterized by vacA typing by PCR and DNA sequencing. Eighty-four of the 87 isolates were s1a/m1, one was s1b/m1, and two could not be typed. Moreover, all isolates in this study were cagA positive. There were no distinct differences between the cytotoxin-producing strains and cytotoxin-nonproducing strains within the 0.73-kb middle region. Japanese strains were highly homologous, with more than 96% identity in this region, in which maximum divergence has been reported. In addition, there were no associations between the specific vacA types and the level of in vitro cytotoxin activity or the clinical consequences. These results indicate that the cagA-positive, s1a/m1-type strains are common in Japan, regardless of the vacA phenotype or clinical outcome.
| S-EPMC229827 | biostudies-other
Project description:Background/aimsDifferences in the Helicobacter pylori infection rate are not sufficient to clarify the dissimilarity of gastric cancer incidence between Myanmar and its neighboring countries. To better understand this trend, the H. pylori virulence gene cagA was characterized in Myanmar.MethodsGlutamate-proline-isoleucine-tyrosine-alanine (EPIYA) patterns and CagA multimerization (CM) motifs of cagA genotypes were examined by performing polymerase chain reactions and DNA sequencing.ResultsOf 69 tested H. pylori strains, cagA-positive patients had significantly more severe histological scores in their antrum than cagA-negative patients. Sequence analysis revealed that 94.1% of strains had Western-type cagA containing an EPIYA motif (92.6%) or EPIYT motif (6.4%). The intestinal metaplasia scores in the antral of patients infected with the ABC and ABCC types of cagA were significantly higher than those of patients with AB-type cagA. Interestingly, in patients infected with H. pylori, 46.3% of strains with three EPIYA motifs contained two identical Western-typical CM motifs, and these patients showed significantly higher antrum inflammation scores than patients infected with two identical nontypical-CM motif strains (p=0.02).ConclusionsIn Myanmarese strains, Western-type cagA was predominant. The presence of CM motifs and the proportion of multiple EPIYA-C segments might partially explain the intermediate gastric cancer risk found in Myanmar.
| S-EPMC5753684 | biostudies-literature
Project description:The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.
| S-EPMC3815135 | biostudies-literature
Project description:Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.
| S-EPMC3154945 | biostudies-literature
Project description:Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.
| S-EPMC3190882 | biostudies-literature
Project description: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 motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial-mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-?B and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA.
| S-EPMC4303776 | biostudies-literature
Project description:Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively contribute to the progression of multistep gastric carcinogenesis.
| S-EPMC2755941 | biostudies-literature
Project description:Helicobacter pylori infection and consumption of a high-salt diet are each associated with an increased risk for the development of gastric cancer. To investigate potential synergism between these factors, we used a global proteomic approach to analyze H. pylori strains cultured in media containing varying salt concentrations. Among the differentially expressed proteins identified, CagA exhibited the greatest increase in expression in response to high salt concentrations. Analysis of 36 H. pylori strains isolated from patients in two regions of Colombia with differing incidences of gastric cancer revealed marked differences among strains in salt-responsive CagA expression. Sequence analysis of the cagA promoter region in these strains revealed a DNA motif (TAATGA) that was present in either one or two copies. Salt-induced upregulation of CagA expression was detected more commonly in strains containing two copies of the TAATGA motif than in strains containing one copy. Mutagenesis experiments confirmed that two copies of the TAATGA motif are required for salt-induced upregulation of CagA expression. In summary, there is considerable heterogeneity among H. pylori strains in salt-regulated CagA expression, and these differences are attributable to variation in a specific DNA motif upstream of the cagA transcriptional start site.
| S-EPMC3418733 | biostudies-literature