VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

transmission. Furthermore, bacteria-invertebrate interactions obviously havecontributed to the evolution of microbial strategies to colonize eukaryotichosts, and to withstand their immune system.The model organisms Galleria mellonella (the greater waxmoth) andManduca sexta (the tobacco hornworm) have been used to decipher geneticdeterminants that play a role in the interaction of Y. enterocolitica withinvertebrates. The most prominent factor identified to be essential forinsecticidal activity is a toxin complex comprising the components TcaA,TcaB, TcaC and TccC. The latter one is biologically active upon ADPribosylatingof actin and RhoA. The expression of TcaA is regulated in atemperature-dependent manner and requires a novel autoregulated LysR-likeinductor. Using the nematode Caenorhabditis elegans for infection with Y.enterocolitica, we demonstrated that a successful exploitation of this hostrequires multiple activities including adhesion, colonization, proliferation,toxin release and subsequent bioconversion. Genome comparison revealed alarge set of factors that are assumed to specifically contribute to these steps.[1] Spanier, B. et al(2010): Yersinia enterocolitica infection and tcaA-dependent killing ofCaenorhabditis elegans. Appl. Environ. Microbiol., in press.[2] Fuchs, T. M. et al (2008): Insecticidal genes of Yersinia spp.: taxonomical distribution,contribution to toxicity towards Galleria mellonella, and evolution. BMC Microbiology 8:214.[3] Heermann, R. and T. M. Fuchs (2008): Comparative analysis of the Photorhabdus luminescensand the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity.BMC Genomics 9:40.[4] Bresolin, G. et al (2006): Low temperature-induced insecticidal activity of Yersinia enterocolitica.Mol. Microbiol. 59, 503-512.consist of a membrane protein complex and an extracellular needle both thatform a continuous channel. Regulated secretion of virulence factors requiresthe presence of SipD at the TTSS needle tip in S. typhimurium. Recently,binding of SipD with bile salts present in the gut was shown to impedebacterial infection.We showed recently that the TTSS needle protomer refolds spontaneously toextend the needle from the distal end. We developed a functional mutant ofthe needle protomer from Shigella flexneri and Salmonella typhimurium tostudy its assembly in vitro. We show that the protomer partially refolds froma-helix into ß-strand conformation to form the TTSS needle. We alsoanalyzed three-dimensional structures of individual SipD, bound to theneedle subunit PrgI, and of the SipD:PrgI in complex with the bile saltdeoxycholate. Structures provide insight to the open state of the tip of theTTSS needle. Five copies each of the needle subunit PrgI and SipD form theTTSS needle tip complex. Assembly of the complex involves majorconformational changes in SipD. The TTSS needle tip complex bindsdeoxycholate with micromolar affinity by a cleft formed at the SipD:PrgIinterface as shown by isothermal titration calorimetry and crystal structureanalysis. In the structure based three-dimensional model of the TTSS needletip the bound deoxycholate is facing the host membrane. Therefore, bindingof bile salts to the SipD:PrgI interface could/may control the TTSS function.Take together our study reveals the molecular assembly mechanisms and thestructure of the TTSS at atomic level.MPV014Pseudomonas aeruginosa virulence analyzed in aDictyostelium discoideum model of infectionA. Zimmermann 1 , B. Nuori 1 , A. Neidig 2 , S. Häußler 1 , C. Matz 1 ,J. Overhage* 21 Cell Biology, Helmholtz Center for Infection Research, Braunschweig,Germany2 Microbiology of Natural and Technical Interfaces, Karlsruhe Institut ofTechnology (KIT), Eggenstein-Leopoldshafen, GermanyPseudomonas aeruginosa is a major opportunistic human pathogen whichproduces a large variety of secreted and cell-associated virulence factors.Since P. aeruginosa infections are difficult to treat due to the emergence ofhighly antibiotic resistant strains, alternative drug targets including virulencefactors are currently being under investigation. Recently, it has been shownthat P. aeruginosa uses similar virulence factors when infecting mammaliansystems or non-mammalian hosts like the social amoeba Dictyosteliumdiscoideum, the nematode Caenorhabditis elegans or the fruit flyDrosophila melanogaster (1,2). In this study, we used a comprehensive P.aeruginosa PA14 transposon mutant library to screen for mutants withreduced virulence towards D. discoideum. A total of 198 P. aeruginosaPA14 transposon mutants were identified to have decreased virulence in thishost model system. In addition to mutants with insertions in the type IIIsecretion system (TTSS), we identified genes involved in type IV pilibiosynthesis and function, PQS production, tryptophan synthesis, transport,central and amino acid metabolism and gene regulation including quorumsensing, global regulators and two-component regulatory systems. FACSanalyses using a gfp-exoT reporter construct revealed reduced TTSS activityin several studied mutants including type IV pili, PQS biosynthesis andcbrAB, a two-component regulatory system involved in nitrogen and carbonmetabolism. Microarray analyses were performed to gain a deeper insightinto the interaction of P. aeruginosa with D. discoideum.1) Hilbi et al., 2007. Environmental Microbiology 9:563-575.2) Alibaud et al., 2008. Cellular Microbiology 10:729-740.MPV015Will be presented as poster with the ID MPP066!MPV017The Zwitterionic Cell Wall Teichoic Acid ofStaphylococcus aureus Provokes Skin Abscesses in Miceby a Novel CD4+ T-Cell-Dependent MechanismC. WeidenmaierInstitute of Microbiology and Infection Medicine (IMIT), Eberhard-Karls-University, Tübingen, GermanyS. aureus is responsible for serious and life-threatening human infections,such as bacteremia, pneumonia, and endocarditis. However the mostprominent S. aureus infections are skin and soft-tissue infections (SSTIs). Incontrast to other types of infections, the microbial factors involved in thepathogenesis of skin infections provoked by S. aureus and the underlyinghost response mechanisms have yet to be studied in detail. Therefore, acomprehensive understanding of the molecular events taking place duringthe course of a staphylococcal skin infection remains largely elusive.Recently, the dogma of adaptive immune system activation was challengedby studies that demonstrated the ability of certain microbial zwitterionicpolysaccharides to be processed and presented via the MHC II pathwaymuch like peptide antigens [1]. Cell wall teichoic acid (WTA) of S. aureus isa zwitterionic polymer, and we demonstrate that purified WTA is able tostimulate CD4+ T-cell proliferation in an MHC II-dependent manner [2].We show in both in vitro and in vivo experiments that the zwitterioniccharge of WTA is crucial for this activity. The results of T cell transferexperiments and CD4+ T cell deficient mouse studies clearly demonstratethat T cell activation by WTA in S. aureus infected tissue stronglymodulates abscess formation. The primary effector cytokine produced byWTA activated T cells is IFN-y which is responsible for promoting the earlyphases of abscess formation. The later stages of abscess progression andclearance rely on a Th17 type response, indicated by high IL-17 levels in theabscess tissues at late timepoints. Our study is both novel and highlyimportant for understanding the molecular basis of the complex pathology ofstaphylococcal SSTIs. In addition, it provides unique insight on the role ofstaphylococcal glycopolymers in bacterial virulence, emphasizing theimportance of investigating these surface molecules from a new perspective.[1] Mazmanian, S. K. and D. L. Kasper (2006): The love-hate relationship between bacterialpolysaccharides and the host immune system. Nat Rev Immunol 6:849-58.[2] Weidenmaier, C. et al (2010): The Zwitterionic Cell Wall Teichoic Acid of Staphylococcus aureusProvokes Skin Abscesses in Mice by a Novel CD4+ T-Cell-Dependent Mechanism. PLoS One 5.MPV016Structure/Function Analysis of the Type 3 SecretionSystem from Salmonella typhimuriumM. Kolbe*, M. LunelliCellular Microbiology, Max Planck Institute for Infection Biology, Berlin,GermanyMany infectious Gram-negative bacteria require a Type Three SecretionSystem (TTSS) to translocate virulence factors into host cells. The TTSSspektrum | Tagungsband 2011