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.

Helicobacter pylori CagA protein induces factors involved in the epithelial to mesenchymal transition (EMT) in infected gastric epithelial cells in an EPIYA‐ phosphorylation‐dependent manner

“…MMP-3 protein expression in total cell lysates, as well as in culture supernatants (representing the secreted form), was found to be dependent on CagA phosphorylation in EPIYA-C repeats. Similarly, the higher the number of EPIYA-C repeats in the expressed CagA protein, the greater the MMP-3 transcriptional activation observed, in line with our previous observations of induction of a more intense scattering and elongation phenotype in the presence of an increasing number of EPIYA-C repeats [43]. Similar results with respect to increased MMP-3 expression were observed when we infected the human gastric adenocarcinoma cell line MKN45 with bacterial strains expressing CagA protein with functional EPIYA-C motifs, although endogenous expression of MMP-3 was also detected under uninfected conditions in this cell type.…”

“…It is widely accepted that, after adhesion of H. pylori to gastric epithelial cells, the bacterial oncoprotein CagA is translocated through the bacterial type IV secretion system, and after intracellular phosphorylation by mammalian kinases on EPIYA C-terminal phosphorylation motifs, induces the appearance of a scattering and elongation phenotype [8,9,15,[43][44][45][46][47][48][49]. Hierarchical phosphorylation of CagA by Src and Abl kinases on repetitive EPIYA sequences located at the C-terminus of the protein, plays a pivotal role in this phenotypic change [8,9,47,50].…”

“…Hierarchical phosphorylation of CagA by Src and Abl kinases on repetitive EPIYA sequences located at the C-terminus of the protein, plays a pivotal role in this phenotypic change [8,9,47,50]. In non-synchronized infection systems involving gastric epithelial cell lines, the percentage of cells adopting the phenotype appears to be dependent on the number of EPIYA-C repeats [8,15,43,44,[46][47][48][49]. This particular scattering and elongation phenotype, termed hummingbird, has been suggested to resemble the epithelial to mesenchymal transition [10,47,49].…”

Inflammation, Immunity, Vaccines for Helicobacter pylori infection

“…Whether the CagA protein itself plays a role in direct activation of NF-jB and other inflammatory pathways is still debated. Kang et al [9] did report direct activation of NF-jB by CagA, and similarly, Papadakos et al [10] demonstrated a novel IL-8 upregulation and secretion that was dependent on the presence of the terminal CagA EPIYA motif [10]. However, the observed effects of CagA were rather small.…”

Genes de patogenicidade de Helicobacter pylori, polimorfismos de citocinas e fatores ambientais afetam o desenvolvimento de doenças gástricas: uma visão geral

“…39 This occurs because the presence of EPIYA-C segments of the CagA protein appears to significantly contribute to the transcriptional activation of IL-8, through activation of NF-kB (factor nuclear kappa B). 40 IL-8 plays a crucial role by chemo attracting and activating neutrophils to the site of infected gastric mucosa. 41 Batista et al showed that increasing of the number of segments EPIYA-C was associated with precancerous gastric lesions and with decreased serum levels of pepsinogen I, which reflects the functional and morphological status of the gastric mucosa.…”