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

105chemotaxis and cytokine induction are crucial for infections, the molecularbasis of the recognition by leucocytes has remained un known. Here wedemonstrate that the human formyl peptide receptor 2 (FPR2) senses S.epidermidis PSMs at nanomolar concentrations. Specific blocking of FPR2or the down regulation of the PSM genes in the agr mutant led to severelydiminished capacities of neutrophils to detect S. epidermidis PSMs.Moreover, Staphylococci developed the quorum sensing system agr tocontrol their detection via human FPR2. Thus, the innate immune systemuses a global mechanism to detect bacterial pathogens. Targeting FPR2may help to manage severe infections induced by different pathogens.Kretschmer D, Nikola N, Dürr M, Otto M and Peschel A,The virulence regulator Agr controls thestaphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2, J Innate Immun.2011 Nov 8Kretschmer D, Gleske AK, Rautenberg M, Wang R, Koberle M, Bohn E, Schoneberg T, Rabiet MJ, BoulayF, Klebanoff SJ, van Kessel KA, van Strijp JA, Otto M, Peschel A: Human formyl peptide receptor 2 senseshighly pathogenic staphylococcus aureus. Cell Host Microbe 2010;7:463-473.Prat C, Bestebroer J, de Haas CJ, van Strijp JA, van Kessel KP: A new staphylococcal anti-inflammatoryprotein that antagonizes the formyl peptide receptor-like 1. J Immunol 2006;177:8017-8026.Wang R, Braughton KR, Kretschmer D, Bach TH, Queck SY, Li M, Kennedy AD, Dorward DW,Klebanoff SJ, Peschel A, DeLeo FR, Otto M: Identification of novel cytolytic peptides as key virulencedeterminants for community-associated mrsa. Nat Med 2007;13:1510-1514.Vuong C, Durr M, Carmody AB, Peschel A, Klebanoff SJ, Otto M: Regulated expression of pathogenassociatedmolecular pattern molecules in staphylococcus epidermidis: Quorum-sensing determines proinflammatorycapacity and production of phenol-soluble modulins. Cell Microbiol 2004;6:753-759.MPV009Salmonella Typhimurium Stimulated TranscriptionalResponse Aids Intracellular ReplicationS. Hannemann*, J.E. GalánYale University School of Medicine, Section of Microbial Pathogenesis,New Haven, United StatesBacterial products are recognized by innate immune receptors leading toinflammatory responses that can both control pathogen spread and result inpathology. Intestinal epithelial cells, which are constantly exposed tobacterial products, prevent signaling through innate immune receptors toavoid pathology. However, enteric pathogens such as SalmonellaTyphimurium, are able to stimulate intestinal inflammation in order topromote bacterial infection. We found that S. Typhimurium can stimulateinnate immune responses in cultured epithelial cells by mechanisms thatdo not involve receptors of the innate immune system. By delivering a setof effector proteins including SopB, SopE and SopE2 through its type IIIsecretion system, the bacterium directly activates Rho-family GTPases thatsubsequently trigger a profound transcriptional reprogramming of hostepithelial cells. These modifications support bacterial replication bymodifying the intracellular environment.MPV010Recruitment of PI3 kinase to caveolin 1 determines the switchfrom the extracellular to the disseminating stage of gonococcalinfectionM. Faulstich* 1 , J.-P. Böttcher 2 , T. Meyer 2 , M. Fraunholz 1 , T. Rudel 11 University of Würzburg Biocenter, Chair of Microbiology, Würzburg, Germany2 Max Planck Institute for Infection Biology, Dept. Molecular Biology,Berlin, GermanyNeisseria gonorrhoeae causes mainly local infections but occasionallyinvades the blood stream thereby initiating disseminating gonococcalinfections (DGI). Gonococcal type 4 pili (T4P) stabilize local infections bymediating microcolony formation and inducing anti-invasive signals.Outer membrane porin PorB IA, in contrast, is associated with DGI andfacilitates the efficient invasion of gonococci into host cells. PorB IA bindsto the scavenger receptor expressed on endothelial cells (SREC1) underlow phosphate conditions, as found e.g. in the vascular system. Here wedemonstrate that both, T4P-mediated inhibition of invasion and PorB IAtriggeredinvasion utilize lipid rafts and signaling pathways that depend onphosphorylation of caveolin-1 at Tyr 14 (Cav1-pY14). We identified the p85regulatory subunit of PI3 kinase (PI3K) and phospholipase C gamma1(PLC1) as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorB IA-induced invasion. Active PI3K induces theuptake of gonococci via a novel invasion pathway involving protein kinaseC and Rac1. Thus the SREC-I/PorB IA interaction triggers a novel route ofbacterial entry into epithelial cells and offers first mechanistic insight intothe switch from local to disseminating gonococcal infection.MPV011Systems biology of the pathogenic bacterium YersiniapseudotuberculosisR. Bücker* 1 , J. Becker 1 , A.K. Heroven 2 , P. Dersch 2 , C. Wittmann 11 Technical University Braunschweig, Institute of BiochemicalEngineering, Braunschweig, Germany2 Helmholtz Center for Infection Research, Department of MolecularInfection Biology, Braunschweig, Germanysteadily working antibiotics. A promising approach to find new targets andtherapeutics is the achievement of a better understanding of the in vivo linkbetween pathogenicity and metabolism in the underlying pathogens. Oneof the relevant microorganisms in this field is Yersinia pseudotuberculosis,the causative agent of self-limiting enteritis, diarrhoea, mesentericlymphadenitis or autoimmune disorders [2]. Concerning the invasion ofmammalian cells, Yersinia is known to have a complex regulatory networkwhich is controlled by nutritional and environmental conditions [3].Here, we investigate its metabolism on the level of molecular in vivofluxes, using state of art 13 C metabolic flux analysis, that is, a coretechnology from industrial biotechnology to perform system-wide pathwayanalysis and subsequent design-based strain optimization [4], so far rarelyfound in the medical field. As starting point for the comprehensiveanalysis, a computational model of the metabolism of Y.pseudotuberculosis was created on basis of available genomic informationand implemented into the flux software platform OpenFlux [5]. Combinedwith 13 C isotope experiments, the model allows to quantify all majorpathways from central carbon metabolism including glycolysis, pentosephosphate pathway, TCA cycle, anaplerotic pathways as well as anabolismof extracellular product formation.Using this novel approach, several mutants of Y. pseudotuberculosislacking specific virulence factors are compared to the wild type to studythe influence of the corresponding genes on metabolism. Simultaneouslyperformed transcriptome profiling provides the link to the layers ofregulation, superimposing the flux network. In further studies the influenceof different antibiotic classes in sub-inhibitory concentrations will beunraveled as well as the effects of temperature, a key parameter during theinfection cycle of Y. pseudotuberculosis.[1]Piddock LJ.: Lancet Infect Dis. 2011 Nov 17.[2] Heroven, A.K. and Dersch, P. (2006): Molecular Microbiology 62(5), 1469-1483.[3] Heroven, A.K. et al (2008): Molecular Microbiology 68(5), 1179-1195.[4] Wittmann, C. (2010): Advances in Biochemical Engineering/Biotechnology 120, 21-49.[5] Quek, L.E. et al (2009): Microbial Cell Factories 8:25.AcknowledgementsThe authors acknowledge financial support by German Research Foundation within the PriorityProgram „Wirtsadaptierter Metabolismus von bakteriellen Infektionserregern (SPP 1316)”MPV012Shigella IpaD has a dual role in type III secretion systemactivationA.D. Roehrich* 1 , E. Guillossou 1 , R.B. Sessions 2 , A.J. Blocker 1,2 , I. Martinez-Argudo 1,31 University of Bristol, School of Cellular and Molecular Medicine, Bristol, Spain2 University of Bristol, School of Biochemistry, Bristol, United Kingdom3 Universidad de Castilla-La Mancha, Facultad de Ciencias Ambientales yBioquímica, Toledo, SpainType III secretion systems (T3SS) are protein injection devices used byGram negative bacteria to manipulate eukaryotic cells. In Shigella, theT3SS is assembled when the environmental conditions are appropriate forinvasion. However, secretion is only activated after physical contact of theneedle tip with the host cell generates an activation signal. The signal istransmitted to the cytoplasm where it triggers secretion. First, translocatorsare secreted which form a pore in the host cell membrane. Second, effectorproteins are translocated into the host cell.The activation process is controlled by components both at the needle tipand in the cytoplasm: At the needle tip, IpaD provides a scaffold for thetranslocators IpaB and IpaC. In its absence no needle tip is formed, theT3SS secretes constitutively and is unable to sense host cell contact. In thecytoplasm, MxiC acts as a gate-keeper of the T3SS. In its absence, thesecretion of pore-forming proteins is decreased and effector proteins are leaked.Questions: What is the role of the major needle tip protein IpaD insecretion activation at the needle tip and in the cytoplasm? What is the roleof the cytoplasmic gate-keeper protein MxiC in translocator secretion?Methods: We have performed random and site-directed mutagenesis ofipaD and mxiC, respectively, and analysed the type III secretion profiles,needle tip composition and host cell interactions of the mutants. We havealso used protein copurification to analyse protein complexes.Results: Random mutagenesis of ipaD identified two classes of mutants.Class I mutants are affected in signal transduction from the needle tipwhile Class II are affected in regulation of ordered secretion inductionfrom the cytoplasm. Site-directed mutagenesis identified a negatively chargedpatch on the surface of MxiC that might be involved in interaction with IpaD.Conclusions: Our data confirms and extends our understanding of theinvolvement of the major needle tip protein in secretion activation andadds a completely novel aspect to the present model for prevention ofpremature secretion, in the absence of an activation signal, from within thebacterial cytoplasm.The rising problem of antimicrobial resistance combined with the shortageof antibacterial drug discovery [1] will result in a decreasing number ofBIOspektrum | Tagungsband 2012