Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ.

Abstract

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) represents a family of important transcription factors in innate immunity. We have previously reported that the gastric pathogen Helicobacter pylori needs the actin-binding protein cortactin for efficient interleukin-8 (IL-8) secretion, which requires NF-κB activation. However, it remained unknown, which exact cortactin signaling mechanism contributes to IL-8 release. In fact, H. pylori profoundly activates NF-κB in wild-type AGS gastric epithelial cells by the effector molecule adenosine diphosphate (ADP)-β-d-manno-heptose (ADPH) in a type IV secretion system-dependent manner. However, the injected CagA protein might contribute to NF-κB activation. The ADPH-stimulated canonical NF-κB cascade involves alpha-kinase 1 and adapter protein TRAF-interacting protein with forkhead-associated domain (TIFA) to activate inhibitor of kappa B (IκB) kinases (IKKs), followed by phosphorylation-dependent degradation of IκBα and subsequent nuclear translocation of p65 NF-κB and IL-8 release. Here, we show that infection of cortactin knockout cells leads to reduced activation of focal adhesion kinase (FAK) and c-Sarcoma (Src) kinase resulting in diminished phosphorylation of IKKβ at tyrosine residue 199 and subsequently phosphorylation of p65 at serine residue 536, both of which are associated with downregulated NF-κB activity. Our results were further supported using FAK and TIFA knockout cells and treatments with purified ADPH and overexpression of CagA, showing cumulative effects in wild-type, but not in knockout cells. These data demonstrate that ADPH-dependent NF-κB activation and IL-8 secretion are enhanced by CagA. Together, we present here a novel CagA>cortactin>FAK>Src>IKKβ signaling cascade, contributing to proinflammatory responses by H. pylori.