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

104pathogenicity of NDM- and non-NDM-carrying strains. However, therapywith imipenem reduced the mortality of larvae infected with non-NDMstrains significantly. In contrast, imipenem injection did not lowermortality rates of larvae infected with NDM strains.Conclusion: The majority of imipenem-resistant strains carried the OXA-23 gene. The NDM-1 gene could already be detected in isolates from2007, two years before NDM-1 was initially discovered. The first NDMmutant, NDM-2, was discovered in an isolate from 2010. With the use ofGalleria mellonella, we established an infection model for evaluatingpathogenicity and antibiotic treatment.MPV005Intimin and invasin export their C-terminus to the bacterialcell surface using an inverse mechanism compared to classicalautotransportP. Oberhettinger* 1 , M. Schütz 1 , J. Leo 2 , D. Linke 2 , I. Autenrieth 11 Institute of Microbiology and Infection Medicine, Medical Microbiology,Tübingen, Germany2 Max Planck Institute for Developmental Microbiology, Protein Evolution,Tübingen, GermanyA large group of bacterial surface proteins is represented by the family ofautotransporter proteins, which belong to the type V secretion system andare found in almost all Gram-negative bacteria. Autotransporter proteinsare often important virulence factors and consist of three functionaldomains: a N-terminal signal sequence, a C-terminal translocator domainand a passenger domain in between. After synthesis in the cytosol and Secmediatedtransport across the inner membrane into the periplasm, thetranslocator domain forms a beta-barrel pore in the outer membrane,presumably with the help of the Bam complex. Through this pore, thepassenger domain is then translocated to the surface of the cell.A second, unrelated family of outer membrane proteins that exposepassenger domains on the bacterial outer surface are the intimins andinvasins, nonfimbrial adhesins from pathogenic bacteria, whichspecifically interact with host cell surface receptors and mediate bacterialattachment or invasion. They are integrated into the bacterial outermembrane with the amino-terminal region, while the carboxy-terminalregion of the polypeptide is exposed on the bacterial outer membrane.Whereas the surface-localized parts of the protein are functionally welldescribed, the topology and insertion of the N-terminal membrane domainand the translocation process have not been described. To investigate thetopology and the mechanism of translocation in more detail, we had acloser look at the amino acid sequence of invasin and intimin usingSignalP, PsiBLAST and HHAlign. Herefrom we got a prediction of thesignal peptide and twelve different -strands, which might build up the -barrel within the outer membrane. Out of these predictions, we developedtopology models of the membrane anchor of invasin and intimin. Byintroduction of HA-Tags defining the orientation of the translocatordomain within the outer membrane and extensive immunofluorescencestudies, we were able to confirm our models. Furthermore, we show thatthe major periplasmic chaperone involved in invasin biogenesis is SurAand that DegP is responsible for quality control if invasin.Moreover, we came to believe that intimin (Int) and invasin (Inv), twomajor pathogenicity factors ofE.coliandYersinia, are monomericautotransporters, with the remarkable difference that their domain order isreversed.MPV006Analysis of the interaction of invasive M1 Streptococcuspyogenes with human endothelial cellsA. Grützner*, M. Rohde, G.S. Chhatwal, S.R. TalayHelmholtz Centre for Infection Research, Medical Microbiology,Braunschweig, GermanyQuestion: Streptococcus pyogenes (GAS) is a human pathogen that causesa variety of diseases ranging from superficial infections to severe invasivediseases like necrotizing fasciitis and streptococcal toxic shock likesyndrome. Serotype M1 and M3 GAS are most frequently associated withinvasive diseases. The M1 surface protein is known to be a major epithelialcell invasin and causes vascular leakage in an animal model. However, tocause an invasive disease, the pathogen has to reach deeper tissue, afterovercoming the endothelial cell barrier. The project focuses on theinteraction of invasive serotype M1 S. pyogenes with human endothelialcells (EC). The aim is to identify possible pathogen- and host cellassociatedfactors that mediate barrier crossing to elucidate the underlyingsignaling cascades in ECs.Methods and Results: Using an in vitro EC infection model we couldshow that different M1 GAS clinical isolates are able to invade polarizedconfluent EC monolayers. After 3 hours of infection streptococci colocalizewith the marker protein Lamp-1, indicating streptococcaltrafficking into the late endosomal/ lysosomal compartment. To testwhether streptococci containing phagosomes also fuse with terminallysosomes, these were pre- loaded with BSA-gold particles and analysedby transmission electron microscopy (TEM). Microscopic images reveal aclose association of streptococci with gold particles, indicating fusion ofthe streptococci containing vacuole with terminal lysosomes. Using anisogenic M1 knock out mutant we demonstrate that the M1 protein is anessential factor for the invasion process into ECs. Furthermore, the entry ofM1 GAS could be significantly reduced using antibodies purified fromrabbit M1 antiserum. Infection studies using M1- coated latex beadssuggest that the M1 protein is not only essential but also can solelymediate entry into ECs. M1- coated latex beads, just like the M1 wt strain,traffic into the late endosomal/ lysosomal compartment.Conclusion: Serotype M1 GAS have the potential to invade polarizedhuman ECs. The M1 surface protein is the EC invasin of M1 GAS. Thus,M1 protein is the second streptococcal factor identified to allow accessinto polarized confluent ECs, one of the strongest cellular barriers in thehuman body.MPV007Lipopolysaccharides of Gram-negative bacteria contribute tothe creation of heparin-induced thrombocytopenia-elicitingantibodies by binding and conformationally altering plateletfactor 4C. Weber* 1 , K. Krauel 2,3 , A. Greinacher 2 , S. Hammerschmidt 11 Ernst Moritz Arndt University of Greifswald, Interfaculty Institute forGenetics and Functional Genomics, Genetics of Microorganisms,Greifswald, Germany2 Ernst Moritz Arndt University of Greifswald, Institute for Immunologyand Transfusion Medicine, Transfusion Medicine, Greifswald, Germany3 Ernst Moritz Arndt University of Greifswald, Center for InnovationCompetence, Humoral Immune Reactions in Cardiovascular Diseases,Greifswald, GermanySome Gram-negative bacteria have been reported to contribute to theetiology of blood clotting disorders, e.g. heparin-inducedthrombocytopenia (HIT) among others. HIT is an IgG-antibody-mediatedadverse drug reaction against complexes of the positively-chargedchemokine platelet factor 4 (PF4) and the most frequently used anionicanticoagulant in clinical medicine, heparin. Interestingly, even heparinnaïvepatients are able to generate IgG-antibodies specific for PF4/heparincomplexes as soon as 4 days after exposure to heparin, presumablybecause these patients have encountered complexes similar to PF4/heparinbefore such as PF4 bound to anionic bacterial surfaces during infections.Likely candidates for negatively charged molecules on the Gram-negativesurface are proteins as well as lipopolysaccharides (LPS).In this study pre-treatment of bacteria with proteinases showed thatproteins play only a minor role in PF4 recruitment to the bacterial surface.However, the components of bacteria binding PF4 have been pinpointedby showing that PF4 interacts with LPS of the Gram-negative modelorganisms Escherichia coli and Salmonella typhimurium. Remarkably, E.coli and S. typhimurium mutants with successively shortened LPSbackbonedisplayed increasing PF4 binding capacity. The highest bindingwas detected in the E. coli waaC and E. coli waaA mutants lackingboth the O-antigens and parts of the core LPS. As the E. coli waaAmutant lacks in addition to the O-antigens and the inner core heptoses alsothe 3-deoxy-D-manno-octulosonic acids (KDO) and as mono-phosphatelipid A showed a decreased binding of PF4, the results suggested that thephosphate groups of lipid A are the actual structures contributing to PF4-binding. Human PF4/heparin antibodies could be affinity-purified frompatient sera using PF4-coated wild-type E. coli as well as PF4-coatedmutants. Thus purified antibodies tested positive in consecutivePF4/heparin ELISA and heparin-induced platelet activation assaysindicated the exposition of PF4/heparin-like epitopes on PF4-coated wildtypeE. coli and waa mutants.Hence, recruitment of PF4 to Gram-negative bacteria via lipid A and itsphosphate groups induces epitopes on PF4 that can trigger a humoralimmune response specific for a wide variety of bacterial species.MPV008Staphylococcus epidermidis and Staphylococcus aureusQuorum Sensing System agr Regulates Formyl PeptideReceptor 2 Ligand Secretion and thereby the Activation of theInnate Immune SystemD. Kretschmer* 1 , N. Nikola 1 , M. Duerr 1 , M. Otto 2 , A. Peschel 11 University of Tübingen, Institut of Medical Microbiology and InfectionMedicine, Tübingen, Germany2 National Institute of Allergy and Infectious Diseases, US NationalInstitutes of Health, Bethesda, MD 20892, United StatesHighly pathogenic Staphylococcus aureus and the opportunistic pathogenStaphylococcus epidermidis secrete phenol-soluble modulin (PSM)peptides.Virulence of S. epidermidis depends mostly on the PSM peptides,which induce chemotaxis in neutrophils and cytokine induction inperipheral blood mononuclearcells (PBMCs). The regulation of PSMsecretion and production occurs through the agr regulator. WhileBIOspektrum | Tagungsband 2012