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

109that CspA can also negatively regulate activation of the alternativepathway independently of its ability to bind CFH and FHL-1.At first, various recombinant CspA proteins with single amino acidsubstitutions have been generated. The mutated proteins were screened fortheir ability to bind CFH and FHL-1 utilizing ligand affinity blotting andELISA. CspA with substitutions at position 101 (CspA K101E) and position242 (CspA D242A) displayed a reduced binding capacity for CFH. Asubstitution at position 246 (CspA L246D) reduced binding of CFH tomarginal levels. By contrast, amino acid substitutions at positions 146(CspA L146H) and 240 (CspA Y240A) abrogated binding of CFH to negligiblelevels. Additionally, proteins CspA L146H and CspA Y240A did not bindsignificant levels of FHL-1 in a ligand affinity blot and CspA L246D showedonly marginal binding of FHL-1.In order to assess whether the mutated CspA proteins, with their reducedor negligible binding capacity for CFH and/or FHL-1, retained theirnegative regulatory effect on the alternative pathway of complement, weemployed a cell-based hemolytic assay. Strikingly, just like the wild-typeCspA, all mutated proteins investigated were able to protect rabbiterythrocytes from complement-mediated lysis in the hemolytic assay.Taken together, our findings indicate that CspA is not only able tonegatively regulate the alternative pathway of complement by usurpinghost regulator molecules such as CFH or FHL-1, but that it can alsoregulate the alternative pathway independently of those molecules. Thechallenge remains, to find out precisely where this second regulatoryactivity in the alternative pathway is targeted.MPV026Zwitterionic cell wall polymers of bacterial pathogensimportantmodulators of T cell dependent infectionsS. Wanner, M. Rautenberg, S. Baur, L. Kull, C. Weidenmaier*IMIT, Medical Microbiology and Hygiene, 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).In contrast to other types of infections, the microbial factors involved inthe pathogenesis of skin infections provoked by S. aureus and theunderlying host response mechanisms have yet to be studied in detail.Therefore, a comprehensive understanding of the molecular events takingplace during the course of a staphylococcal skin infection remains largelyelusive.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. aureusis a 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 in vitro is IFN-, which we found to be responsiblefor promoting the early phases of abscess formation in vivo. The laterstages of abscess progression and clearance rely on a Th17 type response,indicated by high IL-17 levels in the abscess tissues at late time points. Wecurrently try to understand the development and interplay of T cellpopulations after MHCII dependent activation by WTA. In addition wewant to correlate structural differences in WTA polymers of differentGram-positive bacteria to their T cell stimulatory potential. So far wecould demonstrate that the WTA polymer of the skin colonizingcommensal Staphylococcus epidermidis is in contrast to the WTA of S.aureus not able to activate T cells after MHC II presentation. The spacingof the charge centers is shorter in S.epidermidis WTA as compared to S.aureus WTA since S. epidermidis WTA contains glycerol-phosphaterepeating units as opposed to the ribitol-phosphate units of S. aureusWTA. Our study is both novel and highly important for understanding themolecular basis of the complex pathology of staphylococcal SSTIs. Inaddition, it provides unique insight on the role of staphylococcalglycopolymers in bacterial virulence, emphasizing the importance ofinvestigating these surface molecules from a new perspective.1. Weidenmaier C, Peschel A(2008) Teichoic acids and related cell-wall glycopolymers in Gram-positivephysiology and host interactions. Nat Rev Microbiol 6: 276-287.2. Weidenmaier, C., R. M. McLoughlin, and J. C. Lee.2010. The Zwitterionic Cell Wall Teichoic Acid ofStaphylococcus aureus Provokes Skin Abscesses in Mice by a Novel CD4+ T-Cell-Dependent Mechanism.PLoS One5.MPV027Novel murine infection models provide deep insights into the„Ménage à Trois“ of Campylobacter jejuni, microbiota andhost innate immunityM.M. Heimesaat* 1 , A. Fischer 1 , R. Plickert 1 , L.-M. Haag 1 , B. Otto 1 ,A.A. Kühl 2 , J.I. Dashti 3 , A.E. Zautner 3 , M. Munoz 1 , C. Loddenkemper 2 ,U. Groß 3 , U.B. Göbel 1 , S. Bereswill 11 Charité - Universitätsmedizin Berlin, Institut für Mikrobiologie und Hygiene,Berlin, Germany2 Charité - Universitätsmedizin Berlin, Berlin, Germany3 University Medical Center Göttingen, Göttingen, GermanyBackground: Although Campylobacter jejuni-infections have a highprevalence worldwide and represent a significant socioeconomic burden, itis still not well understood how C. jejuni causes intestinal inflammation.Detailed investigation of C. jejuni-mediated intestinal immunopathology ishampered by the lack of appropriate vertebrate models. In particular, micedisplay colonization resistance against this pathogen.Methodology/Principal findings: To overcome these limitations wedeveloped a novel C. jejuni-infection model using gnotobiotic mice inwhich the intestinal flora was eradicated by antibiotic treatment. Theseanimals could then be permanently associated with a complete human(hfa) or murine (mfa) microbiota. After peroral infection C. jejunicolonized the gastrointestinal tract of gnotobiotic and hfa mice for sixweeks whereas mfa mice cleared the pathogen within two days. Strikingly,stable C. jejuni colonization was accompanied by a pro-inflammatoryimmune response indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils and apoptotic cells as well asincreased concentrations of TNF-, IL-6, and MCP-1 in the colon mucosaof hfa mice. Analysis of MyD88 -/- , TRIF -/- , TLR4 -/- , and TLR9 -/- micerevealed that TLR4- and TLR9-signaling was essential forimmunopathology following C. jejuni-infection. Interestingly, C. jejunimutantstrains deficient in formic acid metabolism and perception inducedless intestinal immunopathology compared to the parental strain infection.In summary, the murine gut flora is essential for colonization resistanceagainst C. jejuni and can be overcome by reconstitution of gnotobioticmice with human flora. Detection of C. jejuni-LPS and -CpG-DNA by hostTLR4 and TLR9, respectively, plays a key role in immunopathology.Finally, the host immune response is tightly coupled to bacterial formicacid metabolism and invasion fitness.Conclusion/Significance: We conclude that gnotobiotic and “humanized”mice represent excellent novel C. jejuni-infection and -inflammationmodels and provide deep insights into the immunological and molecularinterplays between C. jejuni, microbiota and innate immunity in humancampylobacteriosis.MPP001Detoxification of nitric oxide during Salmonella pathogenesisA. Arkenberg*, D. Richardson, G. RowleyUniversity of East Anglia, School of Biological Sciences, Norwich, UnitedKingdomThe detoxification of nitric oxide plays a major role during thepathogenesis of Salmonella species. Nitric oxide has bactericidalproperties and is generated by inducible nitric oxide synthase insidemacrophages, where Salmonella is able to reside [1]. Salmonella hasevolved detoxification mechanisms, which allow survival in such astressful environment. Previous work has revealed the importance ofNorV, NrfA and HmpA [2], but more mechanisms are likely to beinvolved. Microarray analysis of Salmonella enterica serovarTyphimurium (S.Typhimurium) highlighted the up-regulation of genes thatcontain putative tellurite resistance domains. The effect of gene deletionshas been investigated using various sensitivity and viability assays rangingfrom exposure to hydrogen peroxide, effects on growth in the presence oftellurite, effect on growth by nitric oxide under oxic and anoxic conditionsto the use of cell culture models.[1] Haraga, A., Ohlson, M. B. and Miller, S. I. (2008) Salmonellae interplay with host cells. Nat RevMicro.6, 53-66[2] Mills, P. C., Rowley, G., Spiro, S., Hinton, J. C. D. and Richardson, D. J. (2008) A combination ofcytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovarTyphimurium against killing by NO in anoxic environments. Microbiology.154, 1218-1228MPP002Discriminative hexaplex PCR strategy for the detection ofmethicillin resistance and virulence factors in StaphylococcusaureusS.M. Shahid*, S.F. Hussain, A. Khatoon, M. Ismail, A. AzharThe Karachi Institute of Biotechnology & Genetic Engineering (KIBGE),University of Karachi, Medical Biotechnology, Karachi, PakistanIn clinical microbiology, phenotypic characterization is laborious and timeconsuming strategy, remains less discriminative among high virulent toless virulent clinically important strains. Induction of moleculartechniques, allow a more accurate and less time consuming way out for theBIOspektrum | Tagungsband 2012