Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

SummaryThe intracellular pathogen Legionella pneumophila activates the transcription factor NF-kB in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kB by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellindeficient (DfliA and DflaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IkBa and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (DdotA; DicmB/dotO), Dot/Icm effectors (DsdbA; DlubX) and two bacterial effector mutants (DenhC; DptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kB activation and intracellular replication and hints at an active interference of NF-kB signalling by L. pneumophila.

Antimicrobial peptides are constituents of the firstline innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human b-defensin 3 (hBD3). We find that hBD3 is highly active against H. pylori in vitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H. pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.

Summary Helicobacter pyloriis a bacterial pathogen that colonizes the gastric niche of ~50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ~3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded co-factor of T4SS function.