Helicobacter pylori type IV secretion system-directed membrane-proximal NF-kB signaling

In the stomach, chronic infection with the pathogen Helicobacter pylori represents a risk factor for the development of chronic inflammation, which is a potent promoter for metaplasia, dysplasia and cancer development. Colonization of gastric epithelial cells by H. pylori induces fast activation of the proinflammatory and survival factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Activation of canonical NF-kB is strictly induced only by H. pylori strains carrying a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS). Further, it has been suggested that Helicobacter outer membrane protein (HopQ), could contribute to NF-kB activation. The detailed mechanism of T4SS-dependent activation of membrane-proximal NF-kB activation is unresolved so far. Regarding the molecular mechanism responsible for canonical NF- B activation and inflammation in infected gastric cancer cell lines we defined as crucial elements the TAK1/TAB complex and the E3 ubiquitin ligase TRAF6, which are situated upstream of the NF-kB inhibitor B kinase (IKK) complex. To identify H. pylori-induced proximal NF-kB signaling molecules which regulate substrate ubiquitinylation, we performed siRNA screens with human ON-TARGETplus siRNA libraries which selectively knockdown F-box and SOCS-box E3 enzymes, or RING-finger and RING-finger-like E3 single protein ligases. Some identified molecules contribute to NF-kB regulation, e.g. Ankyrin repeat and SOCS box protein 3 (ASB3), the Tripartite motif protein containing 28 (TRIM 28) and the ubiquitin-editing enzyme A20. Interestingly, we assigned that H. pylori-induced A20 terminates NF-kB activation, but also attenuates host apoptotic cell death. The overall aim of this project is to decipher the complex regulation of the membrane-proximal signal transmission leading to the activation of canonical NF-kB during H. pylori infection. In detail, we plan to elucidate bacterial T4SS components and Hop-protein adhesins, and their interplay with eukaryotic surface factors (receptors) to unravel NF-kB control in H. pylori infection. Further, a number of evaluated E3 ubiquitin ligases from siRNA screens will be functionally further assessed by a range of established biochemical and cellular approaches regarding their contribution to H. pylori-induced NF-kB activity. Finally, molecular traits of NF-kB signal transmission identified in infected gastric cancer cell lines will be investigated in regard to their in vivo relevance in experimental infection in mice and paraffin embedded human gastric tissue samples from patient biopsies.