VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

PLA activity is directly linked to the hemolytic potential of PlaB. The firstcharacterized member of a new family of lipases also plays an important roleas virulence factor in a guinea pig infection model and is mainly expressedand enzymatically active during the exponential growth phase. Until now,the function of the C-terminal half is unknown, but it contributes to lipolyticactivity. Interestingly, the analysis of three C-terminally truncated versionsof PlaB recombinantly expressed in E. coli revealed, that a lack of only 5amino acids (aa) leads to a 90% decrease of PC-PLA activity. The lack of 10aa at the C-terminus however results in a decrease of 80% PG- and 100% ofPC-PLA activity whereas the removal of 15 aa completely abolishes theenzymatic activity. Furthermore, the purification of sufficient amounts ofsoluble and active PlaB has been pursued for future determination of thecrystal structure, which is helpful for further characterization of PlaBproperties, function and verification of recent data.MPP028Functional expression of truncated Bartonella adhesin A(BadA) in E. coliT. Schmidgen 1 , P. Kaiser 1 , T. Riess 1 , D. Linke 2 , A. Lupas 2 , V. Kempf* 11 Institute for Medical Microbiology and Infection Control, UniversityHospital Frankfuirt am Main, Frankfurt am Main, Germany2 Max Planck-Institute for Developmental Biology, Tübingen, GermanyThe trimeric autotransporter adhesin Bartonella adhesin A (BadA) plays adecisive role in infections with Bartonella henselae. Expression of BadA iscrucial for bacterial autoagglutination, adhesion to host cells, binding toextracellular matrix proteins and the induction of proangiogenicreprogramming via activation of hypoxia inducible factor (HIF)-1. BadA isconstructed modularly consisting of a head domain, a long and repetitiveneck-stalk module and a membrane anchor domain. To analyze the functionof particular BadA domains in detail, the generation of BadA deletionmutants would be highly desirable. However, because of the slow growth ofB. henselae and limited tools for genetic manipulation, we established arecombinant expression system for BadA mutants in E. coli to allowfunctional analysis of certain BadA domains. Therefore, we used (i) atruncated BadA mutant lacking the neck-stalk module (BadA HN23) andexchanged additionally (ii) the BadA HN23 signal sequence with the E. coliOmpA signal sequence, (iii) the BadA HN23 membrane anchor with theYersinia adhesin A (YadA) membrane anchor or (iv) exchanged both ofthese modules. Using a set of expression vectors, these constructs werecloned in several E. coli expression strains to analyze the biologicalfunctions of such BadA-hybrid proteins. Expression of BadA HN23 hybridswas detected via immunoblotting and fluorescence microscopy. All BadAHN23 hybrids were expressed on the surface of E. coli strains althoughquantitative differences were observed. No differences in collagen-bindingbetween E. coli expressing Bad HN23 hybrids and controls were detectable.However, we have preliminary evidence that E. coli expressing BadAhybrid-proteins adheres significantly more to endothelial cells than controlstrains although the total amount of bound bacteria is less than B. henselaewildtype. Further experiments using recombinantly expressed BadA-hybridsshould allow to investigate BadA-mediated bacteria-host cell interactions ingreater detail.MPP029Phenotypic and genotypic characterization ofPseudomonas aeruginosa isolates from technical watersystemsH. Petry-Hansen*, J. Hanke, H.-C. Flemming, J. WingenderBiofilm Center, Department of Aquatic Microbiology, University ofDuisburg-Essen, Essen, GermanyPseudomonas aeruginosa is an opportunistic pathogen that can persist inbiofilms of man-made water systems. P. aeruginosa cells released frombiofilms contaminate the water phase and pose a potential threat to humanhealth.During a period of seven years (2003 - 2010), 77 P. aeruginosa strains wereisolated from water and biofilms of drinking water distribution systems (52),public swimming pools (13), and industrial water systems (12). Clonalrelationship, colony morphology, pigment production, hemolysis, cellsurface hydrophobicity, biofilm formation, resistance against antibiotics andoccurrence of virulence genes of these isolates and additional seven P.aeruginosa reference strains were analysed in order to reveal possiblecorrelations between water source and genotypic as well as phenotypiccharacteristics.Genetic diversity assessed by pulsed field gel electrophoresis of SpeIrestricted genomic DNA of the 77 environmental strains was high; theybelonged to 42 different clonal variants. 64% of these 77 strains and theseven reference strains showed the typical colony morphology for P.aeruginosa, 92.8 % produced at least one of the pigments pyocyanin,pyoverdin, pyorubin and pyomelanin, 98.8 % displayed b-hemolysis andwere sensitive to antibiotics of four different classes, 90.5 % had ahydrophilic cell surface and all strains were able to form biofilms on abiotic(polystyrene) surfaces. In all 84 strains, the virulence gene lasB was detectedas well as either the virulence genes exoS (63 %) or exoU (38 %); only onestrain contained both exoS and exoU.For the first time an extensive pool of P. aeruginosa isolates from differenttypes of technical water systems were characterized phenotypically andgenotypically. No correlation between phenotypic and genotypic traits, andbetween these characteristics and the origin of the strains were detected. Thephenotypic characteristics and the occurrence of virulence genes seemed tobe distributed in a relatively homogeneous way among the clonally unrelatedstrains from diverse man-made water systems.MPP030Staphylococcus lugdunensis SLUSH peptides - more thanhaemolysinsM. Rautenberg* 1 , J. Hwang-Soo 2 , M. Otto 2 , A. Peschel 11 Institute of Microbiology and Infection Medicine (IMIT), MedicalMicrobiology, Eberhard-Karls-University, Tübingen, Germany2 US National Institutes of Health, National Institute of Allergy andInfectious Diseases, Bethesda, MD, USACoagulase-negative staphylococci (CoNS) are becoming more and moreimportant in nosocomial and community-acquired infections such asbacteremia, nosocomial neonatal sepsis, endocarditis, and meningitis andoften these bacteria are resistant to several antimicrobial agents.Staphylococcus aureus and Staqphylococcus epidermidis have been shownto secrete phenol-soluble modulin peptides, which can be sensed bydedicated receptors of the innate immune system and lead to neutrophilresponses such as chemotaxis, calium ion flux, and IL-8 release. To furtherelucidate the ability of neutrophils to sense further CoNS we analyzed theresponse of human primary neutrophils and monocytic cell lines to culturesupernatants from different CoNS species. We found most CoNS species toelicit calcium ion fluxes in leukocytes.One of these CoNS namely S. lugdunensis is outstanding since it behavesmore like S.aureus than other CoNS regarding its virulence and clinicalmanifestation in infections. In fact, this pathogen has often been implicatedin severe inflammatory infections in recent years. These may proceedaggressively and with severity similar to that of S.aureus.S. lugdunensis secretes three small peptides, SLUSH-A, SLUSH-B, andSLUSH-C, which exhibit synergistic haemolytic activity with S.aureus. Inorder to characterize whether the elevated virulence of S. lugdunensis islinked to the presence of these peptides we examinined the response ofhuman neutrophils, monocytes and monocytic cell lines to synthetic SLUSHpeptides.Due to its enhanced virulence S. lugdunensis is not a typical CoNS speciesand deserves more attention regarding its interaction with the adaptiveimmune system.MPP031Role for the cysteine, histidine-dependentamidohydrolase/peptidase (CHAP) domain of theStaphylococcus aureus autolysin/adhesin Aaa inadherence to fibrinogen, fibronectin, and humanendothelial cellsN. Hirschhausen, G. Peters, C. Heilmann*Institute of Medical Microbiology, University Hospital of Münster, Münster,GermanyQuestion: Staphylococcus aureus is a frequent cause of serious and lifethreateninginfections, such as endocarditis, osteomyelitis, pneumoniae, andsepsis. Its adherence to various host structures is considered crucial for theestablishment of diseases. Adherence may be mediated by a variety ofadhesins, among them the autolysin/adhesins Atl and Aaa. Aaa possessesthree N-terminal repeated sequences homologous to a lysine motif (LysM)spektrum | Tagungsband 2011