• Achieving 100% Typeability of Pseudomonas aeruginosa by Pulsed-Field Gel Electrophoresis

      Römling, Ute; Tümmler, Burkhard (American Society for Microbiology, 2000-01)
    • The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

      Pistor, S; Chakraborty, T; Niebuhr, K; Domann, E; Wehland, J (1994-02-15)
    • Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

      Lommel, Silvia; Benesch, Stefanie; Rottner, Klemens; Franz, Thomas; Wehland, Jürgen; Kühn, Ralf (Oxford University Press, 2001-09-15)
    • Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein.

      Schreiber, Kerstin; Boes, Nelli; Eschbach, Martin; Jaensch, Lothar; Wehland, Juergen; Bjarnsholt, Thomas; Givskov, Michael; Hentzer, Morten; Schobert, Max (2006-01-01)
      Recently, we identified a pyruvate fermentation pathway in Pseudomonas aeruginosa sustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol. 186:4596-4604, 2004). Anaerobic long-term survival of P. aeruginosa might be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis of P. aeruginosa cells during a 7-day period of pyruvate fermentation revealed the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter activity was detected in the deeper layers of a P. aeruginosa biofilm using a PPA3309-gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria.
    • Analysis of Cd14 as a genetic modifier of experimental inflammatory bowel disease (IBD) in mice.

      de Buhr, Maike F; Hedrich, Hans-J; Westendorf, Astrid M; Obermeier, Florian; Hofmann, Claudia; Zschemisch, Nils-H; Buer, Jan; Bumann, Dirk; Goyert, Sanna M; Bleich, Andre; et al. (2009-07-27)
      BACKGROUND AND AIM:: By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBir (C3/J)-Il10(-/-) mice [carrying IBD-resistance alleles at this QTL (Cdcs6)] and C57BL/6J (B6)-Il10(-/-) mice, corroborating studies that showed an association of a CD14-promoter polymorphism with Crohn's disease and ulcerative colitis. The aim of the present study was to analyze the molecular mechanisms leading to differential intestinal expression of Cd14 and its contribution to IBD development. METHODS:: Intestinal CD14 expression was assessed by FACS, immunohistochemistry, and ELISA on supernatants of primary epithelial cell and tissue cultures. RAW264.7 cells were stimulated with LPS and PGN in the presence or absence of CD14. Cd14 alleles were sequenced and promoters cloned for luciferase assays in transfected RAW264.7 cells. The severity of typhlocolitis between Cd14(-/-) and wild-type mice was compared in 2 distinct mouse models of IBD (acute DSS and Il10(-/-)). RESULTS:: In the gut, CD14 was detected mainly in its soluble form (sCD14), with higher expression in C3/J-Il10(-/-) mice. Polymorphisms in C3/J mice caused higher activity of the Cd14 promoter (luciferase assays). Intestinal sCD14 concentrations influenced the LPS and PGN responses of RAW264.7 cells. In vivo, genetic deletion of Cd14 aggravated colitis in both mouse models of IBD. CONCLUSIONS:: Our study shows that Cd14-promoter polymorphisms affect CD14 expression and confirms the protective effect of CD14 against experimental IBD, potentially mediated by TLR2- and TLR4-dependent effects on intestinal barrier function. These findings support the concept that human CD14-promoter polymorphisms contribute to disease development. Inflamm Bowel Dis 2009.
    • The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins.

      Veldhoen, Marc; Hirota, Keiji; Westendorf, Astrid M; Buer, Jan; Dumoutier, Laure; Renauld, Jean-Christophe; Stockinger, Brigitta; Division of Molecular Immunology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK. (2008-05-01)
      The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor best known for mediating the toxicity of dioxin. Environmental factors are believed to contribute to the increased prevalence of autoimmune diseases, many of which are due to the activity of T(H)17 T cells, a new helper T-cell subset characterized by the production of the cytokine IL-17. Here we show that in the CD4+ T-cell lineage of mice AHR expression is restricted to the T(H)17 cell subset and its ligation results in the production of the T(H)17 cytokine interleukin (IL)-22. AHR is also expressed in human T(H)17 cells. Activation of AHR by a high-affinity ligand during T(H)17 cell development markedly increases the proportion of T(H)17 T cells and their production of cytokines. CD4+ T cells from AHR-deficient mice can develop T(H)17 cell responses, but when confronted with AHR ligand fail to produce IL-22 and do not show enhanced T(H)17 cell development. AHR activation during induction of experimental autoimmune encephalomyelitis causes accelerated onset and increased pathology in wild-type mice, but not AHR-deficient mice. AHR ligands may therefore represent co-factors in the development of autoimmune diseases.
    • Characterization of the Drosophila lipid droplet subproteome.

      Beller, Mathias; Riedel, Dietmar; Jänsch, Lothar; Dieterich, Guido; Wehland, Jürgen; Jäckle, Herbert; Kühnlein, Ronald P (2006-06-01)
      Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.
    • Contribution of Ena/VASP Proteins to Intracellular Motility of Listeria Requires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

      Geese, Marcus; Loureiro, Joseph J.; Bear, James E.; Wehland, Jürgen; Gertler, Frank B.; Sechi, Antonio S.; Drubin, David (The American Society for Cell Biology, 2002-07)
    • Enzymes Involved in Anaerobic Respiration Appear To Play a Role in Actinobacillus pleuropneumoniae Virulence

      Jacobsen, Ilse; Hennig-Pauka, Isabel; Baltes, Nina; Trost, Matthias; Gerlach, Gerald-F. (American Society for Microbiology, 2005-01)
    • Evaluation of the E test for the assessment of synergy of antibiotic combinations against multiresistant Pseudomonas aeruginosa isolates from cystic fibrosis patients.

      Balke, B; Hogardt, M; Schmoldt, S; Hoy, L; Weissbrodt, H; Häussler, S (2006-01-01)
      The determination of synergistic effects of antimicrobial drug combinations can lead to improved therapeutic options in the antibiotic treatment of cystic fibrosis patients who are chronically infected with multiresistant Pseudomonas aeruginosa isolates. The aim of this study was to evaluate the performance of the E test versus the standard agar dilution checkerboard susceptibility test in the assessment of synergy and, in addition, to determine the activity of two antimicrobial combinations against 163 multiresistant P. aeruginosa isolates from cystic fibrosis patients. The agreement between the checkerboard method and the E test was excellent (>90%) for nonmucoid as well as mucoid isolates from cystic fibrosis patients. The rate of synergy was higher for the antibiotic combination of ceftazidime and tobramycin (28.8% of the cystic fibrosis strains) than for the combination of meropenem and tobramycin (19.0%). However, the probability of synergy for the second antibiotic combination increased significantly when the synergy of the first antibiotic combination had already been demonstrated (Fischer's exact test, p=0.049). The results show that the E test is a valuable and practical method for routine microbiological diagnostics and can aid in the selection of improved antibiotic options in the treatment of cystic fibrosis patients chronically infected with P. aeruginosa.
    • Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection.

      Rakhimova, Elza; Munder, Antje; Wiehlmann, Lutz; Bredenbruch, Florian; Tümmler, Burkhard; Clinical Research Group, OE6710, Hanover Medical School, Hanover, Germany. (2008)
      Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called 'dissociative behaviour'. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of-function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild.
    • Hierarchical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium.

      Kader, Abdul; Simm, Roger; Gerstel, Ulrich; Morr, Michael; Römling, Ute; Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden. (2006-05)
      GGDEF and EAL domain proteins are involved in the turnover of the novel secondary messenger cyclic-di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. In this work the role of the 12 GGDEF domain proteins encoded by the Salmonella enterica serovar Typhimurium (S. Typhimurium) chromosome in rdar morphotype development was investigated. Previously, it was shown that the GGDEF domain protein AdrA activated the biosynthesis of cellulose by production of c-di-GMP. Enhancement of the c-di-GMP levels by overexpression of the GGDEF domain protein AdrA did lead to the activation of curli fimbriae biosynthesis through the elevated expression of CsgD and CsgA. Although knock-out of the chromosomal copy of adrA influenced CsgA expression, CsgD expression was not altered, although more than half of the total cellular c-di-GMP was produced by AdrA at 16 h of growth. On the other hand, chromosomally encoded GGDEF-EAL domain proteins STM2123 and STM3388 were required to additively activate CsgD expression on a transcriptional and post-transcriptional level. Enhanced c-di-GMP levels did overcome temperature regulation of rdar morphotype expression by activation of curli fimbriae as well as cellulose biosynthesis through CsgD expression. Thus in the regulatory cascade leading to rdar morphotype expression c-di-GMP activates several subsequent steps in the network.
    • The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

      Altun, Mikael; Walter, Thomas S; Kramer, Holger B; Herr, Patrick; Iphöfer, Alexander; Boström, Johan; David, Yael; Komsany, Alia; Ternette, Nicola; Navon, Ami; et al. (2015)
      Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.
    • In vitro field exposition of skin cells between 100 GHz and 2.52 THz

      KLeine-Ostmann, Thomas; Jastrow, Christian; Salhi, Mohamed Amine; Schrader, Thorsten; Hintzsche, Henning; Stopper, Helga; Kärst, Uwe; Heinen, B.; Baaske, Kai; Koch, M.; et al. (IEEE, 2010)
    • ISPa20 advances the individual evolution of Pseudomonas aeruginosa clone C subclone C13 strains isolated from cystic fibrosis patients by insertional mutagenesis and genomic rearrangements.

      Kresse, Andreas U; Blöcker, Helmut; Römling, Ute; Research Group Clonal Variability, Division of Cell- and Immune Biology, GBF - German Research Centre for Biotechnology, Mascheroder Weg 1, 38124, Braunschweig, Germany. (2006-05)
      Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.
    • Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues.

      Fleissner, Diana; Hansen, Wiebke; Geffers, Robert; Buer, Jan; Westendorf, Astrid M; Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany. (2010)
      BACKGROUND: In contrast to intestinal CD4(+) regulatory T cells (T(regs)), the generation and function of immunomodulatory intestinal CD8(+) T cells is less well defined. To dissect the immunologic mechanisms of CD8(+) T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied. METHODOLOGY AND PRINCIPAL FINDINGS: HA-specific CD8(+) T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3(+) and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8(+)Foxp3(+) T cells. Antigen-experienced CD8(+) T cells in this transgenic mouse model suppressed the proliferation of CD8(+) and CD4(+) T cells in vitro. Gene expression analysis of suppressive HA-specific CD8(+) T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4(+) T(reg) subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8(+)Foxp3(+) T cells. CONCLUSION AND SIGNIFICANCE: We demonstrate that gut specific antigen presentation is sufficient to induce CD8(+) T(regs)in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.
    • Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense.

      Matz, Carsten; Webb, Jeremy S; Schupp, Peter J; Phang, Shui Yen; Penesyan, Anahit; Egan, Suhelen; Steinberg, Peter; Kjelleberg, Staffan; School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney, Australia. (2008)
      Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.