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

107MPV017Complex c-di-GMP signaling networks mediate the transitionbetween biofilm formation and virulence properties inSalmonella enterica serovar TyphimuriumI. Ahmad 1 , A. Lamprokostopoulou 1 , S. Le Guyon 1 , E. Streck 1 , M. Barthel 2 ,V. Peters 1 , W.-D. Hardt 2 , U. Römling* 11 Karolinska Institutet, Department of Microbiology, Tumor and CellBiology (MTC), Stockholm, Sweden2 ETH Zürich, Institute of Microbiology, D-BIOL, Zürich, SwitzerlandUpon Salmonella enterica serovar Typhimurium infection of the gut, anearly line of defense is the gastrointestinal epithelium which senses thepathogen and intrusion along the epithelial barrier is one of the first eventstowards disease. Recently, we showed that high intracellular amounts ofthe secondary messenger c-di-GMP in S. typhimurium abolishedstimulation of a pro-inflammatory immune response and inhibition ofinvasion of the gastrointestinal epithelial cell line HT-29 suggestingregulation of transition between biofilm formation and virulence by c-di-GMP in the intestine. Here we show that highly complex c-di-GMPsignaling networks consisting of distinct groups of c-di-GMP synthesizingand degrading proteins modulate the virulence phenotypes IL-8production, invasion and in vivo colonization in the streptomycin-treatedmouse model implying a spatial and timely modulation of virulenceproperties in S. typhimurium by c-di-GMP signaling. Inhibition of theinvasion phenotype by c-di-GMP is associated with inhibition of secretionof the type three secretion system effector protein SipA. Inhibition of theinvasion and IL-8 phenotype by c-di-GMP (partially) requires the majorbiofilm activator CsgD and/or BcsA the synthase for the extracellularmatrix component cellulose. Our findings show that c-di-GMP signaling isat least equally important in the regulation of Salmonella-host interactionas in the regulation of biofilm formation at ambient temperature.MPV018Characterization of bacterial strains isolated from communityacquired asymptomatic catheter associated urinary tractinfectionsM. Burghartz*, P. Tielen, R. Neubauer, D. Jahn, M. JahnTU Braunschweig, Institut für Mikrobiologie, Braunschweig, GermanyBacterial colonization of urinary tract catheters is a major cause ofnosocomial infections. Most investigations focus on catheter isolates fromclinical sources. To analyze community acquired catheter infections ofelderly patients seven different bacterial isolates from urinary Foley´scatheters of an urologist practice were identified and characterized withregard to their biofilm formation, urea utilization, DNA degradation andhemolysis activity. For eight antibiotics the minimum inhibitoryconcentrations were determined. Proteus mirabilis, Morganella morganii,Pseudomonas aeruginosa, Alcaligenes faecalis, Enterococcus faecalis,Stenotrophomonas maltophilia and Myroides odoratimimus were isolatedfrom the catheters. All isolates formed biofilms with S. maltophilia and E.faecalis showing the strongest biofilm formation. Urease and DNaseactivity was detected for almost all species. Interestingly, hemolysis wasonly found for P. aeruginosa, S. maltophilia and M. odoratimimus. Onlygentamicin abolished growth on 6 out of seven isolates while kanamycin,ampicillin, nitrofurantoin, tobramycin and cefixime showed almost noeffect. Ciprofloxacin and levofloxacin only inhibited the growth of P.mirabilis and M. morganii. The M. odoratimimus isolate was completelyresistant against all tested antibiotics. We conclude that biofilm formation,urease and DNase production in combination with antibiotic resistance areessential determinants of opportunistic pathogens in community acquiredurinary tract catheter infections.MPV019Global discovery of virulence-associated small RNAs inYersinia pseudotuberculosisB. Waldman 1 , A. K. Heroven 1 , J. Reinkensmeier 2 , J.-P. Schlüter 3 , A. Becker 3 ,R. Giegerich 2 , P. Dersch 11 Abteilung Molekulare Infektionsbiologie, Helmholtz-Zentrum fürInfektionsforschung, Braunschweig;Germany2 Technische Fakultät, Universität Bielefeld, Bielefeld, Germany3 Institut für Biologie, Universität Freiburg, Freiburg, GermanyYersinia pseudotuberculosis is a food-born enteropathogenic bacteriumand closely related to the human pathogen Y. pestis. In both pathogens theRNA chaperon Hfq is required for full virulence (1) indicating that smallRNAs play a crucial role in Yersinia virulence. In fact, we found that in Y.pseudotuberculosis the post-transciptional Csr system participates inmotility, stress resistance and the regulation of virulence genes, e.g. theglobal virulence regulator rovA. RovA controls the expression of earlystage virulence genes, which are important for Y. pseudotuberculosis tocolonize and penetrate the intestinal tract (2). In this study, we used a deepsequencing approach to identify and characterize further so far unknownsRNAs associated with Yersinia virulence.Sequencing of RNA libraries from Y. pseudotuberculosis wildtype and anhfq mutant grown either at 25°C to stationary phase (simulatingenvironmental conditions/early infection phase) or at 37°C to exponentialphase (late infection phase) lead to the identification of 315 putative sRNAout of which 15 were encoded on the Yersinia virulence plasmid pYV. Themajority of these newly identified sRNAs were only found in pathogenicyersiniae. According to the 454 data, one out of four of these newly foundsRNAs is temperature-regulated and about 40% are Hfq-dependent.Expression of selected candidates was further analysed and their influenceon virulence investigated.(1) Schiano CA, Bellows LE, Lathem WW. „The small RNA chaperone Hfq is required for the virulence ofYersinia pseudotuberculosis.“ Infect Immun. 2010 May;78(5):2034-44. Epub 2010 Mar 15.(2) Heroven, AK, Böhme, K., Rohde, M., Dersch, P. „A Csr-type regulatory system, including small noncodingRNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis throughRovM.“ Mol Microbiol. 2008 Jun; 68(5):1179-95.MPV020Fishing for ancient pathogens: A draft genome of a Yersiniapestis strain from the medieval Black DeathV. Schünemann* 1 , K. Bos 2 , H. Poinar 2 , J. Krause 11 University of Tübingen, Institute for Archaeological Sciences, Tübingen,Germany2 McMaster University, Department of Anthropology, Toronto, CanadaThe Black Death is considered to be one of the most devastatingpandemics in human history. Between 1347 and 1352 approximately 30%-50% of Europeans died of this pandemic. Until recently the causativeagent of this epidemic was discussed highly controversial, severalpathogens -Bacillus anthracis, Yersinia pestis or an unknown Filovirusweretaken into account as putative agents. Previous genetic studies wereoften criticized as possible contaminants of modern DNA or closelyrelated soil bacteria. Novel methodical approaches to prove theauthenticity of ancient DNA using characteristic damage patterns enabledus to verify Yersinia pestis as at least one of the causative agents of theBlack Death. For this study 109 samples from skeletal remains of medievalplague victims buried in the East Smithfield cemetery in London wereanalyzed.In the next step 98% of the ancient genome of Y. pestis from four of thevictims was reconstructed to 30-fold genomic coverage. Phylogeneticanalysis revealed that the ancient pathogen is ancestral to most recentplague strains and very close to the root of all genome wide sequenced humanpathogenic Y. pestis strains. These findings indicate that the plague originated asa human pathogen in the late medieval age and suggests that all previous plagueepidemics were caused by an extinct or so far not sequenced branch of Y. pestisor a different pathogen. Furthermore the ancestral Y. pestis strain is highlysimilar to modern human pathogenic strains and therefore weakens theargument that genetic differences contributed to the higher mortality in themedieval era. Other factors beside the microbial genetics, e.g. environmentalchanges, vector dynamics, genetic susceptibility of the host populations or aconcurrent disease, should now be taken into account to explain the observedhigher virulence of the plague during the Black Death pandemic. Thus, the firstgenome of an ancient bacterial pathogen offers a novel opportunity to study theevolution of pathogens.MPV021The YfiBNR signal transduction mechanism reveals noveltargets for the evolution of persistent Pseudomonas aeruginosain cystic fibrosis airwaysT. Jaeger* 1 , J.G. Malone 1,2 , P. Manfredi 1 , A. Dötsch 3 , A. Blanka 4 , S. Häussler 3 ,U. Jenal 11 University of Basel, Biozentrum, Basel, Switzerland2 University of East Anglia, John Innes Centre, Norwich, United Kingdom3 Helmholtz Center for Infection Research, Braunschweig, Germany4 Twincore, Centre of Clinical and Experimental Infection Research, Hannover,GermanyThe genetic adaptation of pathogens in host tissue plays a key role in theestablishment of chronic infections. While whole genome sequencing hasopened up the analysis of genetic changes occurring during long-terminfections, the identification and characterization of adaptive traits is oftenobscured by a lack of knowledge of the underlying molecular processes.Our research addresses the role of Pseudomonas aeruginosa small colonyvariant (SCV) morphotypes in long-term infections. In the lungs of cysticfibrosis patients, the appearance of SCVs correlates with a prolongedpersistence of infection and poor lung function. Formation of P.aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a keyregulator of the SCV phenotype. The effector of this tripartite signalingmodule is the membrane bound diguanylate cyclase YfiN. Through acombination of genetic and biochemical analyses we first outline themechanistic principles of YfiN regulation in detail. In particular, weidentify a number of activating mutations in all three components of theBIOspektrum | Tagungsband 2012