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

108Yfi regulatory system. YfiBNR is shown to function via tightly controlledcompetition between allosteric binding sites on the three Yfi proteins; anovel regulatory mechanism that is apparently widespread amongperiplasmic signaling systems in bacteria. We then show that during longtermlung infections of CF patients, activating mutations invade thepopulation, driving SCV formationin vivo. The identification of mutational“scars” in the yfi genes of clinical isolates suggests that Yfi activity is bothunder positive and negative selection in vivo and that continuousadaptation of the c-di-GMP network contributes to the in vivo fitness of P.aeruginosa during chronic lung infections. These experiments uncover animportant new principle of in vivo persistence, and identify the c-di-GMPnetwork as a valid target for novel anti-infectives directed against chronicinfections.MPV022Methionine sulfoxide reductases defend SalmonellaTyphimurium from oxidative stress and provide bacterialpathogenesisL. Denkel* 1 , S. Horst 1 , S. Fazle Rouf 1 , V. Kitowski 1 , O. Böhm 2 , M. Rhen 3 ,T. Jäger 2 , F.-C. Bange 11 Medical School Hannover, Medical Microbiology and Hospital Epidemiology,Hannover, Germany2 MOLISA GmbH, Magdeburg, Germany3 Karolinska Institute, Microbiology, Tumor and Cellbiology, Stockholm, SwedenQuestion: Oxidative stress produced by the host during Salmonellainfection converts methionine to a mixture of methionine-S-sulfoxide(Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) [1]. The methioninesulfoxide reductases MsrA and MsrB are known to protect bacteria byrepairing oxidized methionine, the former being specific for the S-formand the latter being specific for the R-form [1,2]. In this study wecharacterize MsrA, MsrB and a third methionine sulfoxide reductase,MsrC, in S.Typhimurium.Methods: For this study we generated deletion mutants in S. Typhimuriumusing the one-step inactivation via homologous recombination [3].Phenotypic analyses of S.Typhimurium strains included growthexperiments, challenging bacteria with exogenous H 2O 2, infection ofactivated RAW 264.7 macrophages and competitive infection of Balb/cJmice with S. Typhimurium. For biochemical characterization of MsrA andMsrB the proteins were overexpressed in E. coli, purified and examined byNADPH linked reductase activity assay.Results: Here we show that deletion of msrA in S. Typhimurium increasedsusceptibility to exogenous H 2O 2 and reduced bacterial replication insideactivated macrophages and in mice. In contrast, an msrB mutant showedthe wild type phenotype. We constructed msrB and msrC mutant strainsin a methionine auxotrophic background of S. Typhimurium. The msrCmutant but not the msrB mutant failed to utilize free Met-R-SO.Recombinant MsrA was active against both free and peptidyl Met-S-SO,whereas recombinant MsrB was only weakly active and specific forpeptidyl Met-R-SO. To dissect the role of MsrC in oxidative stressresponse we compared an msrC single mutant and an msrBmsrCdouble mutant, and found that MsrC affects survival of S. Typhimuriumfollowing exposure to H 2O 2, growth in macrophages and in combinationwith MsrB also in mice.Conclusions: Thus in summary, we showed that mutants of S.Typhimurium lacking components of the methionine sulfoxide reductasepathway are attenuated in vitro when exposed to H 2O 2, inside activatedmacrophages and in mice. Previously, MsrA and MsrB were considered tobe the principle enzymes of the msr-system that play a role in oxidativestress response. Here we show that in addition MsrC contributessignificantly to thwart the damage caused by oxidative stress inS.Typhimurium [4].1. Weissbach H, Etienne F, Hoshi T, Heinemann SH, Lowther WT, et al. (2002) Peptide methioninesulfoxide reductase: structure, mechanism of action, and biological function. Arch Biochem Biophys 397:172-178.2. Boschi-Muller S, Olry A, Antoine M, Branlant G (2005) The enzymology and biochemistry ofmethionine sulfoxide reductases. Biochim Biophys Acta 1703:231-238.3. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. Proc Natl Acad Sci U S A 97:6640-66454. Denkel LA, Horst SA, Rouf SF, Kitowski V, Böhm OM, Rhen M, Jäger T, Bange F-C (2011) MethionineSulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium. PLoS ONE 6(11): e26974.doi:10.1371/journal.pone.0026974MPV023RNase Y of Staphylococcus aureus does not result in bulkmRNA decay but in activation of virulence genesG. Marincola* 1 , T. Schäfer 2 , K. Ohlsen 2 , C. Goerke 1 , C. Wolz 11 Interfaculty Institute of Microbiology and Infection Medicine, Tübingen,Germany2 Institute for Molecular Infection Biology, Würzburg, GermanyBacteria are able to cope with environmental changes by rapidly alteringmRNA expression. Coordinated RNA decay is also essential to allowquick adjustment of RNA levels. Several RNases are involved in RNAdecay, processing and maturation of the different RNA species. Sequencehomologues of major Escherichia coli enzymes cannot be identified infirmicutes. Recently, an essential endoribonuclease, RNase Y, wasidentified in Bacillus subtilis as a key member of the degradosome andproposed to be important for bulk mRNA turnover. Here we analyzed therole of RNase Y homologue rny in the human pathogen Staphylococcusaureus. In contrast to B. subtilis, rny is obviously not essential in S. aureussince rny deletion mutants could readily be obtained. As a model forRNase Y action, we used the processing of saePQRS operon coding forcomponents of a global virulence regulatory system. The most prominenttranscript of this operon was shown to be generated by specificendonucleolytic cleavage of a larger autoregulated transcript. In rnymutants the saePQRS processing was no more detectable. To gain insightinto the expression of genes affected by RNase Y, gene expressionprofiling between rny mutant and wild type was compared throughmicroarray analysis. As expected for an RNase mutant, the mRNA levelsof several genes/operons were significantly increased in the rny mutant.Accordingly, the half life of one of these operons was shown to beextended from 1.1 to 12.7 min. However, the half-lifes of other mRNAspecies, including virulence genes and regulators such as agr, were notsignificantly altered in the rny mutant. This suggests that in S. aureusRNAse Y does not lead to decay of bulk RNA but rather influence mRNAexpression in a tightly controlled regulatory manner. Interestingly, therewere many genes down-regulated in the rny mutant. Among those genes,which are presumably controlled by RNase Y in an indirect way, we couldidentify various known to be involved in the pathogenesis of S. aureus.The promoter activities of those virulence genes (e.g. hlg and spa) were indeedseverely impaired in the rny mutants. RNase Y was moreover required for fullvirulence in a murine S. aureus bacteremia model. In summary, in S. aureusRNase Y is essential for coordinated activation of virulence genes but does notlead to bulk RNA decay as shown in B. subtilis.MPV024sarA negatively regulates Staphylococcus epidermidis biofilmformation by modulating expression of 1 MDa extracellularmatrix binding protein and autolysis dependent release ofeDNAM. Christner, M. Busch, C. Heinze, M. Kotasinska, G. Franke, H. Rohde*UKE Hamburg-Eppendorf, Medizinische Mikrobiologie, Hamburg, GermanyBiofilm formation is essential for Staphylococcus epidermidispathogenicity in implant-associated infections. Nonetheless, largeproportions of invasive S. epidermidis isolates fail to show accumulativebiofilm growth in vitro. We here tested the hypothesis that this apparentparadox is related to the existence of superimposed regulatory systemssuppressing a multi-cellular biofilm life style. Transposon mutagenesis ofclinical significant but biofilm negative S. epidermidis 1585 was used toisolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chiefregulator of staphylococcal virulence. Genetic analysis found thatinactivation of sarA induced biofilm formation via over-expression ofgiant 1 MDa extracellular matrix binding protein (Embp), serving as anintercellular adhesin. In addition to Embp, augmented extracellular DNA(eDNA) release significantly contributed to biofilm formation in mutant1585sarA. Increased eDNA amounts indirectly resulted from upregulationof metalloprotease SepA, leading to boosted processing ofmajor autolysin AtlE, in turn resulting in augmented autolysis and releaseof chromosomal DNA . Hence, this study identifies sarA as a negativeregulator of Embp- and eDNA dependent biofilm formation, linking SepAmediatedescape from defensin dermicidine with biofilm related protectionfrom phagocytosis. Our data establish a central role of sarA as a regulatorensuring S. epidermidis adaptation to hostile environments.MPV025CspA of Borrelia burgdorferi is a regulator of the alternativepathwayA. Koenigs* 1 , P. Kraiczy 1 , C. Siegel 1 , S. Früh 1 , T. Hallström 2 , C. Skerka 2 ,P.F. Zipfel 2,31 University Hospital Frankfurt, Medical Microbiology and InfectionControl, Frankfurt, Germany2 Leibniz Institute for Natural Product Research and Infection Biology,Department of Infection Biology, Jena, Germany3 Friedrich Schiller University, Jena, GermanyThe Lyme disease spirochete, Borrelia burgdorferi, is transmitted to thehuman host through the bite of an infected tick. Upon entry into thebloodstream the spirochetes are immediately confronted by the host’sinnate immune system. The complement system is an integral part ofinnate immunity and in order to establish a persistent infection in the host,Borreliae have evolved a number of sophisticated means to evadecomplement-mediated killing.The outer surface protein CspA of B. burgdorferi contributes tocomplement resistance by binding host complement regulators such asfactor H (CFH) and factor H-like protein-1 (FHL-1). Here we demonstrateBIOspektrum | Tagungsband 2012