Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells

Abstract

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor‐like (‘hummingbird’) phenotype. In this study, using a cagA mutant complemented with wild‐type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti‐phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA–green fluorescent protein (GFP) fusion proteins and two‐dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C‐terminus. Site‐directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).

The Helicobacter pylori cytotoxin CagA is essential for suppressing host heat shock protein expression

“…1b). The 'hummingbird' effect has previously been shown to be dependent upon an intact cagPAI and phosphorylation of translocated CagA (Backert et al 2001;Segal et al 1999). Consistent with this, the P12ΔcagA mutant did not induce 'hummingbird' morphology in infected AGS cells (Fig.…”

“…Wild-type H. pylori strain P12 (P12wt) and its CagAdeficient isogenic mutant (P12ΔcagA) were used to infect the gastric adenocarcinoma cell line AGS to model H. pylori infection of gastric epithelia in vitro (Backert et al 2001;Kwok et al 2002;Kwok et al 2007). Following 24 h of infection, HSP expression was quantitated by Western blot analysis.…”

Gastric Ulcer Healing – Role of Serum Response Factor

“…CagA is one of the cag PAI encoded genes. Upon attaching to the mucosal epithelial cells, cag PAI secretion system transports CagA into the host cells, causing actin cytoskeleton rearrangement into "Hummingbird" phenotype (Backert et al, 2001). These studies link SRF to the main cause of peptic ulcer.…”

Assessment of the mosaic structure in the Helicobacter pylori cagA gene 3′-region using an improved polymerase chain reaction–based assay

“…Previous studies have shown that the CagA cytotoxin is directly injected into epithelial cells via a type IV secretion system, encoded by genes located in the cag -pathogenicity island (cag-PAI) [1,4,5]. In the host cell, CagA localises to the inner surface of the plasma membrane and undergoes phosphorylation on specific tyrosine residues within repeating penta amino acid Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, and in some cases Glu-Pro-Ile-Tyr-Thr (EPIYT), present at the C-terminus of the protein [2,6,7]. Tyrosine phosphorylation is carried out by host cell kinases such as Abl and Src [6,8,9].…”

“…In the host cell, CagA localises to the inner surface of the plasma membrane and undergoes phosphorylation on specific tyrosine residues within repeating penta amino acid Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, and in some cases Glu-Pro-Ile-Tyr-Thr (EPIYT), present at the C-terminus of the protein [2,6,7]. Tyrosine phosphorylation is carried out by host cell kinases such as Abl and Src [6,8,9]. The tyrosine phosphorylated CagA binds to a cytoplasmatic Src Homology 2 (SH2) domain of Src Homology 2 phosphatase 2 (SHP-2).…”