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

121characteristics. These studies should contribute to a better understanding ofthe fundamental biology of these medically important pathogenic fungi.Staib P, Morschhäuser J (2005) Differential expression of theNRG1repressor controls speciesspecificregulation of chlamydospore development inCandida albicansandCandida dubliniensis.Mol Microbiol 55: 637-652MPP050The immune modulatory zwitterionic cell wall polymer ofStaphylococcus aureus - an important role in CA-MRSApathogenicity?S. Wanner*, M. Rautenberg, C. WeidenmaierInstitute of Microbiology and Infection Medicine (IMIT), MedicalMicrobiology, Tuebingen, GermanyStaphylococcus aureus is a major pathogen, in both nosocomial andcommunity-acquired infections that can cause a large variety of infectionsbut skin and soft-tissue infections (SSTIs) are the most common typecaused by CA-MRSA. The pathogenicity of CA-MRSA strains seems todepend on an array of different virulence factors; however the relativeactivity of these factors is still unclear. Recently, we demonstrated that thecell wall polymer WTA (wall teichoic acid) of S. aureus is a majormodulator for the early phase of abscess formation [1]. The immunemodulatory activity of WTA depends on its zwitterionic character and theability to stimulate CD4+ T-cell proliferation in an MHC II-dependentmanner [2], which is in contrast to the current dogma a non peptideantigen. We found that highly pathogenic CA-MRSA strains exhibit anelevated amount of WTA in their cell wall. Purified protein-free cell wallfractions from CA-MRSA induce T-cell proliferation and cytokineproduction more efficiently than cell wall from non CA-MRSA. Thus, cellwall fractions of CA-MRSA strains are more active in skin abscessformation, which can be attributed to the higher WTA amount in their cellwall. Hence, up-regulation of WTA expression is one of the possiblemechanisms CA-MRSA exploit to gain virulence. To confirm ourhypothesis, we currently elucidate a detailed expression profile ofimportant structural genes of WTA biosynthesis by quantitative real-timePCR. Therefore, selected CA-MRSA strains will be compared to differentnon CA-MRSA strains in vitro and ex vivo using a skin abscess model inmice. Furthermore, we plan to measure the expression of teichoic acidbiosynthesis enzymes on the protein level. Our goal is to get more insightsinto the regulatory elements involved in WTA biosynthesis. This studymay contribute to a better understanding of the complex pathology ofSSTIs caused by highly virulent CA-MRSA strains.[1] Weidenmaier, C., R. M. McLoughlin, and J. C. Lee. The Zwitterionic Cell Wall Teichoic Acidof Staphylococcus aureus Provokes Skin Abscesses in Mice by a Novel CD4+ T-Cell DependentMechanism. PLoS One 5. 2010.[2] Mazmanian, S. K., and D. L. Kasper. The love-hate relationship between bacterialpolysaccharides and the host immune system. Nat Rev Immunol 2006. 6:849-58.MPP051Cell contact dependent virulence gene expression in YersiniapseudotuberculosisW. Opitz*, P. DerschHelmholtz Zentrum für Infektionsforschung, Molekulare Mikrobiologie,Braunschweig, GermanyThe enteropathogenic bacterium Yersinia pseudotuberculosis colonizes thehuman gut and transmigrates through the mucosal cell layer intounderlying lymphatic tissues and organs. This causes several gut- andlymph- associated diseases and in rare cases autoimmune diseases.As Y. pseudotuberculosis can be found in the environment as well as insideits host’s body, it needs to perfectly adapt to these particular conditions.Especially virulence genes are tightly environmentally regulated. We showthat Y. pseudotuberculosis senses cell contact to distinguish betweenenvironment and host and to adapt gene expression. Especially genesrequired in the late phase of infection (yop regulon) seem to beupregulated upon contact to human cells.Within this work the impact of cell contact on the expression of the outermembrane protein YadA and its transcriptional regulator LcrF wasinvestigated. During infection YadA mediates adhesion to and invasioninto epithelial cells and helps to evade host’s immune system. Monolayersof epithelial cells (HEp-2) were infected with Y. pseudotuberculosiscarrying yadA and lcrF promoter reporter gene fusions to GFP orluciferase. The expression pattern of bacteria in contact to cells werecompared to free bacteria and analyzed by fluorescence microscopy,luminescence detection or western blotting. We could show that theexpression of yadA is directly activated through a cell contact dependentexpression of its regulator lcrF. Further, CsrA, a RNA-binding protein ofthe carbon storage system, is part of this cell contact sensing cascade. Theimportance of several other factors could be excluded.By analyzing various mutants and performing microarray analysis we wantto identify more participating factors and the cell contact sensor.MPP052Expression of filamentous hemagglutinin in Bartonella henselaeE. Wüstenhagen*, B. Franz, V.A.J. KempfKlinikum der Johann Wolfgang Goethe-Universität, Institut für MedizinischeMikrobiologie und Krankenhaushygiene, Frankfurt am Main, GermanyThe gram-negative, zoonotic pathogen Bartonella henselae causes catscratch disease and vasculoproliverative disorders. In recent years, twoessential pathogenicity factors ofB. henselae have been investigated in detail: the trimeric autotransporteradhesin Bartonella adhesin A (BadA) and the VirB/D4 type IV secretionsystem (VirB/D4 T4SS). Analysis of the genomic sequence of B. henselaegave evidence for an additional pathogenicity factor, the filamentoushemagglutinin (FHA). Eight genes of different length encode homologuesof filamentous hemagglutinin (FhaB), and four genes encode homologuesof FhaC/HecB of Bordetella pertussis forming potentially a two partnersecretion system. Until now, nothing is known on the role of FHA ininfections with B. henselae. Here, we analyzed the expression of fhaB andfhaC/hecB in two different B. henselae strains (Marseille, Hoston-1) underdifferent growth conditions (different pH values, at 30 and 37 °C) byquantitative realtime-RT-PCR. Our data revealed that fhaB and fhaC/hecBwere (i) expressed in both B. henselae strains and (ii) expression was pHdependentmeaning that the expression level increased with increasing pHvalues. Cultivation temperature did not have an effect on expression.These results give first evidence, that filamentous hemagglutinin is in factexpressed in B. henselae and might therefore play a role in Bartonellainfections.MPP053Determination of intracellular survival of Streptococcus agalactiaein the interaction with monocytic and granulocytic cellsA. Sagar* 1 , C. Klemm 1 , S. Mauerer 1 , G. van Zandbergen 2 , B. Spellerberg 11 University of Ulm, Institute of Medical Microbiology and Hospital Hygiene,Ulm, Germany2 Federal Institute of vaccines and bio-medical drug, Immunology, Langen,GermanyStreptococcus agalactiae (Group B Streptococci, GBS) is an importantcause of human invasive infections in newborns, pregnant women andimmunocompromised adult patients. The ß-hemolysin of GBS is a surfaceassociated toxin and regarded as a major virulence factor of GBS. It isregulated by the cov two-component regulatory system, which controlsnumerous virulence factors of GBS. To determine the role of the ß-hemolysin for intracellular survival and to rule out the effect of othervirulence factors controlled by cov, we investigated hemolytic andnonhemolytic GBS mutants for intracellular survival in primary humangranulocytes and THP-1 cells.We examined the role of ß-hemolysin for interaction with the monocyticand granulocytic cells using a serotype Ia S. agalactiae wild type strainand an isogenic nonhemolytic deletion mutant of this strain. Both strainswere fluorescently labeled with an EGFP expressing plasmid. Followinginfection of eukaryotic cells with GBS, the intracellular bacteria wereevaluated by FACS analysis and culturing of intracellular bacteria.Interestingly, the non-hemolytic mutants were able to survive in theintracellular environment in significantly higher numbers than thehemolytic strain. A finding that was observed for primary granulocytes aswell as for THP-1 cells. To exclude the possibility that the observeddifferences in survival were due to host cell death induced by thehemolytic but not the non-hemolytic strain, Lactate Dehydrogenase (LDH)assays were carried out and confirmed a better survival capacity of thenonhemolytic strain. To assess the induction of IL-8 following infectionwith GBS, ELISA determinations were performed. While a considerablerelease of IL-8 could be observed, we could however not find a significantdifference in their ability to induce the chemokine. To determine thebacterial mediators of IL-8 release in this setting, cell wall preparationsfrom both strains were incubated with THP-1 cells. Both preparations werefound to exert a potent proinflammatory stimulus on THP-1 cells. Inconclusion our results indicate, that the S. agalactiaeß-hemolysin has astrong influence on the intracellular survival of GBS and that a tightlycontrolled regulation of ß-hemolysin expression is required for thesuccessful establishment of GBS in different host niches.BIOspektrum | Tagungsband 2012