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

103MPV001The lipodepsipeptide empedopeptin inhibits cell wall biosynthesisthrough Ca 2+ -dependent complex formation with peptidoglycanprecursorsH. Brötz-Oesterhelt 1 , *A. Mueller 2 , D. Muench 2 , Y. Schmidt 3 , K. Reder-Christ 4 ,G. Schiffer 5 , G. Bendas 4 , H. Gross 3 , H.-G. Sahl 2 , T. Schneider 21 University of Duesseldorf, Pharmaceutical Biology, Duesseldorf, Germany2 University of Bonn, Medical Microbiology, Immunology and Parasitology,Bonn, Germany3 University of Bonn, Pharmaceutical Biology, Bonn, Germany4 University of Bonn, Pharmaceutical Chemistry, Bonn, Germany5 AiCuris, Wuppertal, GermanyEmpedopeptin is a natural lipodepsipeptide antibiotic with potentantibacterial activity against multi-resistant Gram-positive bacteriaincluding methicillin-resistant Staphylococcus aureus and penicillinresistantStreptococcus pneumoniae in vitro and in animal models ofbacterial infection. Here, we present its so far elusive mechanism ofantibacterial action.Empedopeptin selectively interferes with late stages of cell wallbiosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetyl-glucosamine incorporation into polymeric peptidoglycan and theaccumulation of the ultimate soluble peptidoglycan precursor UDP-Nacetyl-muramicacid-pentapeptide in the cytoplasm. Using membranepreparations and the complete cascade of purified, recombinant late-stagepeptidoglycan biosynthetic enzymes and their respective purifiedsubstrates, we show that empedopeptin forms complexes withundecaprenyl pyrophosphate containing peptidoglycan precursors. Theprimary physiological target of empedopeptin is undecaprenylpyrophosphate-N-acetylmuramicacid-pentapeptide-N-acetyl-glucosamine(lipid II), which is readily accessible at the outside of the cell and whichforms a complex with the antibiotic in a 1 : 2 molar stoichiometry. Lipid IIis bound in a region that involves at least the pyrophosphate group, thefirst sugar, and the upper parts of stem peptide and undecaprenyl chain.Undecaprenyl pyrophosphate and also teichoic acid precursors are boundwith lower affinity and constitute additional targets. Calcium ions arecrucial for the antibacterial activity of empedopeptin, as they promotestronger interaction with its targets and with negatively chargedphospholipids in the membrane. Based on the high structural similarity ofempedopeptin to the tripropeptins and plusbacins, we propose thismechanism of action for the whole compound class.MPV002The Staphylococcus aureus plasmin-sensitive protein Pls is aglycoproteinI. Bleiziffer 1 , K. McAulay 2 , G. Xia 3 , M. Hussain 1 , G. Pohlentz 4 , A. Peschel 3 ,S.J. Foster 2 , G. Peters 1 , C. Heilmann* 11 University Hospital Münster, Institute for Medical Microbiology, Münster,Germany2 University of Sheffield, Institute of Molecular Microbiology, Sheffield, UnitedKingdom3 University of Tübingen, Medical Microbiology and Hygiene Department,Tübingen, Germany4 University Hospital Münster, Institute of Medical Physics and Biophysics,Münster, GermanyQuestion: Until recently, the inability of bacteria to glycosylate proteinshas been considered a dogma. Now, it is widely accepted that bacteria canglycosylate proteins. Most bacterial glycoproteins identified to date arevirulence factors of pathogenic bacteria, i.e. adhesins and invasins.Methods and Results: To study the impact of protein glycosylation instaphylococci, we analysed lysostaphin lysates of the methicillin-resistantStaphylococcus aureus (MRSA) strain 1061 by SDS-PAGE and PeriodicAcid Schiff stain that specifically stains glycosylated proteins. Wedetected two glycosylated surface proteins with molecular masses of ~270and ~180 kDa, the latter being a degradation product of the 270 kDaprotein and identified as plasmin-sensitive protein Pls by massspectrometry. In a search for potential glycosyltransferases (Gtfs) involvedin the glycosylation of Pls, we expressed the pls gene that is encoded onSCCmec type I in the SA113 wild-type strain and various Gtf mutants(SA113gtfAB, SA113bgt, SA113E3, SA113gtfABE3, SA113gtfABE3E4).All strains, but the SA113gtfAB mutants produced glycosylated versions ofPls indicating a role for GtfA and/or GtfB in Pls glycosylation. However,the MRSA mutant strain COLgtfAB still produced a glycosylated versionof Pls suggesting that MRSA genomes carry additional gtf genes. Blastsearches identified two potential gtf genes downstream of pls, which wetermed gtfC and gtfD. Expression analysis indicated that both, GtfC andGtfD, are involved in glycosylation of Pls in the MRSA strains COL and1061. Moreover, the construction and characterization of pls subclonesrevealed that glycosylation occurs at the C-terminal SD repeats of Pls. Plsis known to prevent S. aureus adherence to fibrinogen and fibronectin andalso its internalization by host cells probably acting by steric hindrance.ELISA adherence and internalization assays indicated that these functionsare not due to the glycosylation of Pls. However, we detected a significantimpact of Pls glycosylation on its binding to peptidoglycan suggesting apotential function in the proper targeting and/or surface display of Pls.Conclusion: The S. aureus plasmin-sensitive protein Pls is a glycoproteinand GtfC/GtfD are Gtfs involved in its glycosylation. Glycosylation of Plshas no impact on its ability to prevent adherence or internalization, butpotentially plays a role in its proper targeting and/or surface display.Currently, further analyses are on the way to determine the impact of sugarmodifications on S. aureus pathogenicity, which may represent promisingnew targets for therapeutic measures.MPV003From target to therapy - Expression and characterization ofan anti-staphylococcal antibodyB. Oesterreich* 1 , R. Kontermann 2 , C. Erck 2,3 , U. Lorenz 2,3,4 , K. Ohlsen 1,2,3,41 Institute of Moleculare Infectionbiology, University Würzburg, Würzburg,Germany2 Institute of Cellbiology, Stuttgart, Germany3 HZI, Braunschweig, Germany4 Department of Surgery, Würzburg, GermanyThe Gram-positive bacteriumStaphylococcus aureusis the major cause ofnosocomial infections. In particular, diseases caused by methicillinresistantS.aureus(MRSA) are associated with higher morbidity, mortalityand medical costs due to showing resistance to several classes ofestablished antibiotics and their ability to develop resistance mechanismsagainst new antibiotics rapidly. Therefore, immunological strategies basedon therapeutic antibodies have the potential to close the gap for an efficienttreatment of MRSA.The focus of our work is theidentification of surface components ofstaphylococci with potential as an immunodominat antigen. In thisregardthe immunodominant staphylococcal antigen A (IsaA) has beenidentified as a putative target for immunotherapy due to its expression byall clinical strainsinvivo, and its surface exposure. Preclinical experimentsrevealed protective properties of a monoclonal mouse anti-IsaA antibody(UK-66)in vitroby phagocytosis assays and in mouse infection models.Therefore, this mouse monoclonal antibody was selected for humanization.The hybridoma clone UK-66 was the basis for the identification of theantigen binding domain against IsaA. The coding sequence was used toconstruct recombinant scFv and scFvFc fragments towards IsaA and tohumanize the murine antigen binding domain. The fragments werecharacterized in their function and specificity by Western Blot analysis,ELISA-studies, immuno-fluorescence analysis and FACS experiments.The results revealed that all constructed fragments posses a highspecificity towards IsaA and the property of the antigen binding fragmentsto detect IsaA on the cell surface of differentS. aureusstrains. After thesestudies the whole antibody was constructed and its function wascharacterized by ELISA-studies, FACS experiments and killing assays.Based on these results the humanized anti-IsaA antibody has the potentialfor a successful immunotherapy against MRSA.MPV004Antibiotic resistance and pathogenicity of NDM-carryingAcinetobacter baumanniiC. Szagunn*, T.A. Wichelhaus, V.A.J. Kempf, S. GöttigJohann Wolfgang Goethe-Universitätsklinikum, Medizinische Mikrobiologieund Krankenhaushygiene, 60596 Frankfurt am Main, GermanyQuestion: The gram-negative bacterium Acinetobacter baumannii causessevere nosocomial infections. The worldwide spread of multidrug resistantA. baumannii is a serious global health threat. Aggravation of antibiotictreatment is mainly caused by OXA-lactamases and NDM (New Delhimetallo-beta-lactamase). In this study, we analyzed the prevalence of thementioned resistance genes in clinical A. baumannii isolates. Furthermore,we examined the role of NDM in an infection model since it is suggestedto be part of a genomic pathogenicity island.Methods: DNA from A. baumannii clinical isolates was screened by PCRfor the presence of NDM and OXA-lactamases and verified by sequencing.Antibiotic susceptibility testing was done using Vitek2 and E-test method.Pathogenicity of NDM- and non-NDM strains was investigated in timekill-kineticsusing the Galleria mellonella (larvae of the Greater WaxMoth) infection model.Results: A. baumannii strains with an extended antibiotic resistanceprofile were isolated from 57 patients from intensive care units between2001 and 2011. We discovered 38 imipenem-resistant strains; amongthose, 19 were positive for OXA-23, 3 for OXA-24 and 9 for OXA-58. Wedetected four NDM-carrying isolates: one NDM-1 positive strain from2007 and two from 2011. In addition, we found one NDM isolate with anovel point mutation from 2010 which is now being considered as NDM-2. NDM-carrying A. baumannii were resistant to all tested antibioticsexcept the reserve antibiotics tigecycline and colistin.Time-kill-kinetics in infection experiments using our newly establishedGalleria mellonella infection model revealed no difference betweenBIOspektrum | Tagungsband 2012