Infected AGS Cells Research Articles

Lactiplantibacillus plantarum ZJ316 Reduces Helicobacter pylori Adhesion and Inflammation by Inhibiting the Expression of Adhesin and Urease Genes.

The present study aims to investigate the anti-Helicobacter pylori (H. pylori) effects of Lactiplantibacillus plantarum ZJ316 (L. plantarum ZJ316) both in vitro and in vivo. This study finds that L. plantarum ZJ316 effectively suppresses H. pylori adhesion in inhibition (Pre-ZJ316), competition (Co-ZJ316), and displacement (Post-ZJ316) assays, and Pre-ZJ316 displaying the most potent inhibitory effect with an impressive inhibition ratio of 70.14%. Upon anti-adhesion, L. plantarum ZJ316 significantly downregulates the expression of H. pylori virulence genes, including ureA, ureB, flaA, and sabA, with inhibition ratios of 46.83%, 24.02%, 21.42%, and 62.38% at 2 h, respectively. In addition, L. plantarum ZJ316 is observed to reduce the level of interleukin 8 (IL-8) and improve cell viability in infected AGS cells. Furthermore, in vivo studies show that supplementation with L. plantarum ZJ316 effectively hinders H. pylori colonization and significantly suppresses the infiltration of immune cells and IL-8 production with H. pylori infection, protecting host from inflammatory damage. L. plantarum ZJ316 exhibits excellent adhesion inhibition on H. pylori, and may be used as a probiotic candidate in the prevention or adjuvant therapy of gastric disease caused by H. pylori.

Antagonistic Activities of Lactobacillus rhamnosus JB3 Against Helicobacter pylori Infection Through Lipid Raft Formation.

Helicobacter pylori is a Gram-negative pathogen that can increase the risk of stomach cancer in infected patients. H. pylori exploits lipid rafts to infect host cells. Infection triggers clustering of Lewis x antigen (Lex) and integrins in lipid rafts to facilitate H. pylori adherence to the gastric epithelium. H. pylori infection can be treated with probiotics containing lactic acid bacteria that offer numerous benefits to the host while lacking the side effects associated with antibiotic therapy. Previously, we showed that the cell-free supernatant (CFS) derived from Lactobacillus rhamnosus JB3 (LR-JB3) at a multiplicity of infection (MOI) of 25 attenuated the pathogenicity of H. pylori. In this study, we established a mucin model to simulate the gastric environment and to further understand the influence of mucin on the pathogenesis of H. pylori. Porcine stomach mucin dramatically upregulated H. pylori virulence gene expression, including that of babA, sabA, fucT, vacA, hp0499, cagA, and cagL, as well as the adhesion and invasion ability of H. pylori and induced increased levels of IL-8 in infected-AGS cells. The CFS derived from LR-JB3 at a MOI of 25 reduced the expression of H. pylori sabA, fucT, and hp0499 in mucin, as well as that of the Lex antigen and the α5β1 integrin in AGS cells during co-cultivation. These inhibitory effects of LR-JB3 also suppressed lipid raft clustering and attenuated Lewis antigen-dependent adherence, type IV secretion system-mediated cell contact, and lipid raft-mediated entry of VacA to host cells. In conclusion, LR-JB3 could affect H. pylori infection through mediating lipid raft formation of the host cells. The currently unknown cues secreted from LR-JB3 are valuable not only for treating H. pylori infection, but also for treating diseases that are also mediated by lipid raft signaling, such as cancer and aging-associated and neurodegenerative conditions.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence

ABSTRACT Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the β-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Effects of Helicobacter pylori on the glutathione-related pathway in gastric epithelial cells

Virulence factors of Helicobacter pylori (H. pylori) are diverse, so various biological responses happen in a host infected with H. pylori. The aim of this study is to conduct the metabolomics-based evaluation on H. pylori infection. AGS human gastric carcinoma cells were infected with H. pylori strain 26695, and then the altered metabolite pathways in the infected AGS cells were analyzed by metabolomics. Metabolites related to the glutathione (GSH) cycle were downregulated by H. pylori infection. Next, we evaluated the effects of H. pylori on the GSH-related pathway in AGS cells infected with H. pylori isolated from patients with atrophic gastritis (AG), duodenal ulcer (DU) and gastric cancer (GC). We found that the declined degree of GSH levels and oxidative stress were greater in AGS cells infected with GC strains than DU and AG-derived strains. There were no significant differences in almost mRNA expressions of GSH-related factors among different clinical strains, but the protein expression of glutathione synthetase was lower in AGS cells infected with GC-derived strains than DU and AG-derived strains. Our data demonstrates that GC-derived H. pylori-induced oxidative stress in a host is stronger and GC-derived strains may have suppressive influences on the host’s GSH-related defense systems.

A Conserved Helicobacter pylori Gene, HP0102, Is Induced Upon Contact With Gastric Cells and Has Multiple Roles in Pathogenicity.

Contact with host cells is recognized as a signal capable of triggering expression of bacterial genes important for host pathogen interaction. Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly upregulated expression of a gene, HP0102, in the adhered bacteria in all strains examined, including several Indian clinical isolates. The gene is highly conserved and ubiquitously present in all 69 sequenced H. pylori genomes at the same genomic locus, as well as in 15 Indian clinical isolates. The gene is associated with 2 distinct phenotypes related to pathogenicity. In AGS cell-adhered H. pylori, it has a role in upregulation of cagA expression from a specific σ(28)-RNAP promoter and consequent induction of the hummingbird phenotype in the infected AGS cells. Furthermore, HP0102 has a role in chemotaxis and a ΔHP0102 mutant exhibited low acid-escape response that might account for the poor colonization efficiency of the mutant.

The inhibitory effect of flavonoids on interleukin-8 release by human gastric adenocarcinoma (AGS) cells infected with cag PAI (+) Helicobacter pylori.

IntroductionIt is well known that the presence of Helicobacter pylori in the stomach induces gastritis and causes an immune response. Exposure of gastric epithelial cell lines to this germ induces the secretion of interleukin-8 (IL-8), which is a potent PMN-activating chemotactic cytokine. Interleukin-8 is usually elevated in gastric biopsy samples of patients with H. pylori-associated gastritis and significantly increases in the supernatant of in vitro cultivated biopsy samples of gastric mucosa with active H. pylori gastritis. Interleukin-8 is an activating factor for leucocytes and other pro-inflammatory factors, free radicals, and proteolytic enzymes. That is why natural compounds potentially useful in therapy are still investigated – among them flavonoids. They reveal anti-oxidative and anti-inflammatory activities and significantly inhibit the gastric mucosa damage.The aim of the studyWas the estimation of the anti-inflammatory effects of flavonoids on H. pylori-induced activation of human gastric adenocarcinoma cells (AGS). After infection of AGS cells by cag PAI (+) H. pylori in vitro, secretion of IL-8, effects of flavonoids on viability of AGS cells, and effects of flavonoids on increase of H. pylori were determined. Such flavones as chrysin, quercetin, kaemferide, flavanone, galangin, and kaempferol were examined.ResultsThis study has shown an inhibitory effect of flavonoids on the release of IL-8 through infected AGS cells (except chrysin), and no toxic effects to AGS cells were observed. Galangin revealed antibacterial effects against H. pylori. Flavonoids limit the inflammatory process through the inhibition of IL-8 release in infected AGS cells with H. pylori. The strongest inhibitor of IL-8 was galangin.

Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells.

BackgroundHyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).ResultsThe growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.ConclusionHigher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.

Su1863 Genome-Wide Identification of Epstein-Barr Virus (EBV) Driven Gastric Cancer-Related DNA Methylation by Medip-Chip Profiling of EBV Infected AGS Cells

Su1863 Genome-Wide Identification of Epstein-Barr Virus (EBV) Driven Gastric Cancer-Related DNA Methylation by Medip-Chip Profiling of EBV Infected AGS Cells

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

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

Phosphoproteomic analysis of differential expression of AGS cellular proteins in response to Helicobanter pyiori infection

Objective To research the differential expression of trace phosphorylated proteins in human gastric adenocarcinoma epithelial (AGS) cells infected by Helicobacter pylori. Methods H. pylori 26695 strain infected AGS cells 4 h and AGS cells was cultivated for 4 h as a comparison. The proteins of AGS and comparison AGS cells were extracted. Their phosphorylated proteins were enriched by metal ion af-finity adsorption enrichment techniques. After desalinated and purified the phosphorylated proteins samples were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Computer assisted image analysis was used to analyze the differential proteomic expression. The significantly differentially ex-pressed proteins were unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF). Results Fifteen kinds of proteins were down-regulated, 4 kinds of new proteins were observed, 1 kind of proteins were up-regulated, 1 kind of proteins unexpression. The 21 proteins that were significantly differentially expressed , including cellular calcium ion homeostasis, transcription, interpretation, protein folding and transport, ribosomal assembly, centrosome replication, chromosome stability, cellular structure, cellular proliferation and apoptosis. Conclusion H. priori can cause a wide range change to human gastric adenocarcinoma epithelial cell protein pheshorylation. This change character has great significance to further comprehensive understanding of the pathogenesis of H. pylori. Key words: Affinity adsorption; Phosphorylated proteins; 2-DE; Pathogenic mechanism

MRNA levels of TLR4 and TLR5 are independent of H pylori

To determine if the presence H pylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels. For the in vivo assays, gastric biopsies were obtained from 40 patients and H pylori status was determined. For the in vitro assays, human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains. H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA. The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells. TLR4 and TLR5 mRNA levels were examined by real-time PCR. The presence of H pylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies. The mRNA levels of both receptors were not influenced by the vacA status (P > 0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P > 0.05). In the in vitro assay, the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAs1 status or the clinical condition associated with the strains (P > 0.05 for both TLR4 and TLR5). The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells, both in vivo and in vitro, are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5.

Helicobacter pylori CagA Containing ITAM‐Like Sequences Localized to Lipid Rafts Negatively Regulates VacA‐Induced Signaling In Vivo

Helicobacter pylori CagA is injected into the host cell and tyrosine-phosphorylated. We examined tyrosine-phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine-phosphorylation sites were determined using quadropolar time-of-flight (Q-TOF) mass spectrometry analysis. Specific anti-pY CagA polyclonal and anti-CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Mass spectrometry identified five crucial tyrosine-phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)-infected AGS cells. CagA protein also had an immuno-receptor tyrosine-based activation motif (ITAM)-like amino acid sequences in the 3' region of the cagA, EPIYATIx27EIYATI, which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX-100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine-phosphorylated GIT1/Cat1 (G protein-coupled receptor kinase-interactor 1/Cool-associated tyrosine-phosphorylated 1), substrate molecules of receptor type protein-tyrosine phosphatase (RPTPzeta/beta), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA-induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine-phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H. pylori-related disease.