• A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.

      Düvel, Juliane; Bertinetti, Daniela; Möller, Stefan; Schwede, Frank; Morr, Michael; Wissing, Josef; Radamm, Lena; Zimmermann, Bastian; Genieser, Hans-Gottfried; Jänsch, Lothar; et al. (2012-02)
      In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.
    • Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa.

      Bielecki, Piotr; Jensen, Vanessa; Schulze, Wiebke; Gödeke, Julia; Strehmel, Janine; Eckweiler, Denitsa; Nicolai, Tanja; Bielecka, Agata; Wille, Thorsten; Gerlach, Roman G; et al. (2015-07-27)
      Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P. aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments.
    • Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection.

      Hatesuer, Bastian; Hoang, Hang Thi Thu; Riese, Peggy; Trittel, Stephanie; Gerhauser, Ingo; Elbahesh, Husni; Geffers, Robert; Wilk, Esther; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNβ after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.
    • Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

      Osterloh, Anke; Kalinke, Ulrich; Weiss, Siegfried; Fleischer, Bernhard; Breloer, Minka; Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany. osterloh@bni.uni-hamburg.de (2007-02-16)
      Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.