VAAM-Jahrestagung 2012 18.–21. März in Tübingen

117MPP033Phosphorylation in Staphylococcus aureus, the role of PknBand StpS. Donat* 1 , P. Francois 2 , T. Schäfer 1 , J. Schrenzel 2 , D. Becher 3 , K. Ohlsen 11 Institute for Molecular Infection Biology, University of Wuerzburg,Wuerzburg, Germany2 Service of Infectious Diseases, Geneva University Hospital, Geneva, Switzerland3 Institute for Microbiology, University of Greifswald, Greifswald, GermanyPosttranslational modification of proteins increases there functionaldiversity. Very important for the function of a variety of enzymes isthereby the switch between an active or inactive status by reversiblephosphorylation. In signal transduction pathways these phosphorylationand dephosphorylation events are a key mechanism to response to intraandintercellular signal.Although prokaryotes were thought to use predominantly two componentsystems for signal transduction, genes encoding serine/threonine, tyrosinekinases and phosphatases have been identified in a wide variety ofmicroorganisms. The aim of our work is the analysis of the function androle of the serine/threonine kinase PknB and its corresponding phosphataseStp in the metabolism and virulence of the major pathogenStaphylococcusaureus. Therefore, we investigated the transcriptomic profile of theS.aureuswild type strain NewmanHG [1] and its isogenic deletion mutantspknB, stpand stp/pknB. By this approach we identified an influenceofpknBandstpon the expression of major virulence regulators as well as oncentral metabolic pathways. Furthermore, by using a proteomic approachsubstrates and interaction partners of this signaling system could beanalyzed. Additionally, pknBshowed a higher virulence potential in an invivo murine infection model whereas stpwas significantly attenuated.Our expression and proteomic data together with the in vivo infectionresults strongly suggest an important role the signal transduction modulPknB and Stp in the metabolism and virulence ofS. aureus.[1] Mainieroet al., 2010MPP034Host cell adhesion and immune evasion of zoonotic and nonzoonoticMRSAP. Jung* 1 , K. Bleses 1 , A. Feßler 2 , J. Blatt 3 , B. Ballhausen 4 , S. Schwarz 2 ,R. Köck 5 , K. Becker 4 , C. Cuny 6 , S. Monecke 7 , R. Ehricht 8 , L. von Müller 1 ,M. Herrmann 1 , M. Bischoff 11 University of Saarland Hospital, Institute of Medical Microbiology andHygiene, Homburg, Germany2 Friedrich-Loeffler-Institute (FLI), Institute of Farm Animal Genetics,Neustadt-Mariensee, Germany3 Färber Emil GmbH, Zweibrücken, Germany4 University Hospital of Münster, Institute of Medical Microbiology, Münster,Germany5 University Hospital of Münster, Institute of Hygiene, Münster, Germany6 Robert Koch Institute, Wernigerode, Germany7 Dresden University of Technology, Institute of Medical Microbiology andHygiene, Dresden, Germany8 Alere Technologies GmbH, Jena, GermanyDuring recent years livestock-associated methicillin-resistantStaphylococcus aureus (LA-MRSA), mostly belonging to the clonalcomplex (CC) 398, have been recognized as a source for human infectionswith a potential for a major healthcare challenge. So far the pathogenicmechanism of endemic LA-MRSA and of their methicllin-susceptiblecounterparts (MSSA) for transmission across species barriers,colonization, and disease formation are mostly unknown. Crucial stepstowards disease formation include bacterial adhesion to host cell matrixcomponents, invasion of cells and tissues, and evasion from the hostimmune response. We are addressing these processes by analyzing theability of epidemiologically relevant zoonotic and non zoonotic MRSAand MSSA to adhere to human and animal cells and cell matrixcomponents that are known to be important for bacterial adhesion. In asecond approach, the uptake of zoonotic and non zoonotic MRSA andMSSA by human and porcine blood phagocytes is investigated.First results indicate a strain-specific ability to adhere to humankeratinocytes, different types of human/bovine collagen and immobilizedhuman/bovine plasma fibronectin. The latter reveals a host dependency ofsome animal isolates. Overall, zoonotic MRSA CC398 isolates displayed amore heterogeneous fibronectin-binding than human hospital-acquiredMRSA isolates. In whole blood phagocytosis assays, we observedsignificant differences in the bacterial uptake by porcine and human bloodgranulocytes, but no such differences with regard to the origin of theisolates.Taken together, our data suggest that MRSA derived from animals cannotbe easily distinguished from those derived from humans by the adhesiveand immune evasive properties investigated.MPP035An FNR-Homologue in the aerobic phytopathgenic bacteriumXanthomonas campestris pv.vesicatoria controls expression ofthe operon encoding a high-affinity Cytochrome d OxidaseJ. Kirchberg* 1 , T. Heinz 2 , T. Hoppe 1 , B. Thiemer 1 , G. Sawers 11 University, Microbiology, Halle, Germany2 University, Biotechnology, Halle, GermanyThe plant pathogen Xanthomonas campestris pv.vesicatoria (Xcv)is anobligate aerobic oxidase-negative - proteobacterium that causes bacterialspot disease on pepper and tomato plants. It grows in the intercellularspace between plant cells where it must overcome iron- and oxygenlimitation,as well as cope with reactive oxygen species (ROS) and nitricoxide, which potentially form part of the host defense mechanism.Analysis of the genome of Xcv revealed a gene (xcv1871) encoding anFNR-like transcription factor, which we refer to as FLP (FNR-likeprotein). In E. coli FNR is an oxygen-responsive transcription regulatorswitching on gene expression under oxygen-limiting or anaerobic growthconditions. FLP from Xcv and E. coli FNR share 50% amino acid sequencesimilarity and FLP could partially complement an E. coli fnr mutant.Notably the N-terminal Cys residues required for [Fe-S] clustercoordination and the C-terminal E-SR motif, which in FNR recognises thesequence TTGAT - N4 - ATCAA, are conserved, suggesting that FLP isalso an oxygen-sensitive transcriptional regulator in this obligate aerobe.The putative FLP-recognition sequence is conserved in front of the cydABoperon encoding a high-affinity cytochrome d oxidase. Growth studiesunder oxygen-limiting conditions demonstrated that Xcv can not grow inthe absence of oxygen; however, RT-PCR analyses revealed thattranscription of the cydAB operon was increased when oxygen levels in thegrowth medium were low. Deletion of the flp gene in Xcv preventedtranscription of the cydAB operon and led to a reduced growth phenotypeof the mutant in pepper plants. Introduction of the flp gene on a plasmidrestored both cydAB expression and growth in planta. Taken together, ourstudies suggest that FLP might be required to allow optimal growth of Xcvunder oxygen-limiting conditions during infection of the host plant.MPP036Icm/Dot-dependent modulation of phagocyte migration byLegionella pneumophilaS. Simon*, M. Wagner*, H. HilbiMax von Pettenkofer-Institut, Ludwig-Maximilians-Universität, München,GermanyLegionella pneumophila, the causative agent of Legionnaires' disease,infects and replicates in macrophages and in free-living amoebae such asAcanthamoeba castellanii or Dictyostelium discoideum [1]. Followinginternalization by a host cell, L. pneumophila forms a "Legionellacontainingvacuole" (LCV), which avoids fusion with lysosomes,communicates with the endoplasmic reticulum and allows intracellularreplication. LCV formation is governed by the bacterial Icm/Dot type IVsecretion system (T4SS) through the secretion of a myriad of “effectorproteins”. Some of these effectors target host phosphoinositide lipids orsmall GTPases and thus subvert eukaryotic vesicle trafficking and signaltransduction pathways. Here we investigate the impact of L. pneumophilainfection on chemotaxis and motility of mammalian and protozoanphagocytes. To this end, an under-agarose cell migration assay wasestablished to monitor the migration behavior of RAW264.7 murinemacrophages and D. discoideum amoebae towards tumor necrosis factor(TNF) or folate, respectively. The motility of phagocytes infected withwild-type L. pneumophila was severely reduced compared to macrophagesor D. discoideum infected with L. pneumophila icm/dot mutant strains,which cannot grow intracellularly. Our results indicate that L. pneumophilaimpairs phagocyte migration in an Icm/Dot-dependent manner andsuggests that some L. pneumophila effector proteins interfere with hostsignaling pathways that are involved in chemotaxis and cell migration.Currently, we characterize in detail the interplay between bacterial andhost factors participating in phagocyte motility.[1] Hilbi, H., C. Hoffmann, and C. F. Harrison, Legionella spp. outdoors: colonization,communication and persistence. Environ Microbiol Rep, 2011.3: p. 286-296.MPP037Crystal structure and regulation mechanisms of the AdenylylCyclase CyaB from the human pathogen Pseudomonas aeruginosaH. Topal* 1,2 , N.B. Fulcher 3 , C. Steegborn 2 , M.C. Wolfgang 31 University Konstanz, Biophysics and Membrane Protein Crystallography,Konstanz, Germany2 University , Biochemistry, Bayreuth, Germany3 University, Cystic Fibrosis/Pulmonary Research and Treatment Center, NorthCarolina, USA, United StatesPseudomonas aeruginosa is a major cause of nosocomial infections, andtreatment of P. aeruginosa infections is hindered by the bacterium’s highantibiotic resistance. The regulatory network controlling P. aeruginosaBIOspektrum | Tagungsband 2012