title: Presence of terminal EPIYA phosphorylation motifs in Helicobacter pylori CagA contributes to IL-8 secretion, irrespective of the number of repeats.
abstract: CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1(-/-) mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection.
authors: Laboratory of Medical Microbiology, Hellenic Pasteur Institute, Athens, Greece.
authors: Konstantinos S
authors: KS
authors: Papadakos
authors: Ioanna S
authors: IS
authors: Sougleri
authors: Andreas F
authors: AF
authors: Mentis
authors: Efstathios
authors: E
authors: Hatziloukas
authors: Dionyssios N
authors: DN
authors: Sgouras
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D005347:Fibroblasts / Q000378:metabolism / Q000382:microbiology; D016480:Helicobacter pylori / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D015533:Transcriptional Activation / Q000187:drug effects; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016209:Interleukin-8; D020930:MAP Kinase Kinase Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016328:NF-kappa B; D051057:Proto-Oncogene Proteins c-akt; D000099151:MAP Kinase Kinase Kinase 7; C080866:cagA protein, Helicobacter pylori