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

123which is then used to exchange the wild type copy of the gene with thetagged gene version.Finally, integrating these isotope and tap-tagged data into bioinformaticapproaches allows us not only to reconstruct fluxes but also the dynamicsof different protein complexes. Consequently, we will determine to whatdegree protein complexes are required for physiological fitness of S.aureus, which accounts to a better understanding of its pathophysiology.MPP058The Patatin-like Protein VipD/ PatA - a phospholipase A ofLegionella pneumophila playing a role in bacterium-host interactionK. Seipel*, P. Aurass, A. FliegerRobert Koch-Institut, FG 11 Division of Bacterial Infections, Wernigerode,GermanyThe phospholipase VipD/ PatA is one of eleven patatin-like proteins (PLP)found in Legionella pneumophila. Patatin-like proteins are lipid-acylhydrolases mainly characterized in plants so far, but they were previouslyshown to be widely coded within bacterial genomes. In L. pneumophila,VipD/ PatA was determined to be a substrate of the type IVB secretionsystem by Shohdy et al. (PNAS 2005). We focused on this protein becauseit is the L. pneumophila PLP which is most similar to ExoU, a potentphospholipase and cytotoxin of Pseudomonas aeruginosa that causes rapidhost cell death upon injection by the type III secretion system of thispathogen. We previously found that similar to ExoU, VipD/ PatA localizesto the cytoplasmic membrane after expression in A549 lung epithelialcells. Here, the C-terminal region of the protein plays an essential role,because deletion of the 129 C-terminal amino acids abolishes propertargeting. We now aimed to characterize the protein determinants fortranslocation of VipD/ PatA to the cytoplasmic membrane. Therefore, wemutated or deleted potential phosphorylation sites, special conservedmotifs and a potential transmembrane domain as well as a region of lowcomplexity to evaluate the influence of these parts for membranelocalization. The lipolytic activity of VipD/ PatA, for which Serin-72embedded in a G-X-S-X-G lipase motif is essential, is not required formembrane targeting.MPP059sRNA-mediated control of the primary invasion factor invasinin Yersinia pseudotuberculosisS. Seekircher*, K. Böhme, A.K. Heroven, W. Opitz, P. DerschHZI, MIBI, Braunschweig, GermanyYersinia pseudotuberculosis is an enteric human pathogen that causes gutassociateddiseases. The primary virulence determinant is the outermembrane protein invasin. This protein mediates bacterial binding to andinvasion through the epithelial cells of the gut.Invasin expression is controlled by rovA (regulator of virulence A) inresponse to the surrounding temperature and ion availability (1). One keyregulator of rovA in turn is the csr (carbon storage regulator) system. It iscomposed of the RNA-binding protein CsrA and two regulatory RNAs,csrB and CsrC. These RNAs sequester CsrA thus controlling its function (2).Expression and stability of the two RNAs is controlled by differentregulators and sensory cascades. Recent findings showed that CsrBexpression is activated upon bacterial contact to epithelial cells. Althougha two-component regulator system is known to induce CsrB synthesis thecell contact signal is not integrated via this sensor system.The regulation of CsrC involves various transcriptional and posttranscriptionalmodulators. For example the Yersinia modulator A (YmoA)confers CsrC RNA stability. However, CsrC stability is not directlymediated by YmoA. Microarray analysis indicated that YmoA affectsexpression of different RNases, which might control CsrC turnover.(1) Nagel G., Lahrz A., Dersch P. „Environmental control of invasin expression in Yersiniapseudotuberculosisis mediated by regulation of RovA, a transcriptional activator of the SlyA/Horfamily.“ Mol Microbiol. 2001 Sep; 41(6):1249-69.(2) Heroven, AK, Böhme, K., Rohde, M., Dersch, P. „A Csr-type regulatory system, includingsmall non-coding RNAs, regulates the global virulence regulator RovAofYersiniapseudotuberculosisthrough RovM.“ Mol Microbiol. 2008 Jun; 68(5):1179-95.MPP060Proteomic characterization of host pathogen interactionduring internalization of S. aureus by A549 cellsK. Surmann* 1 , M. Simon 1 , P. Hildebrandt 1 , H. Pförtner 1 , V.M. Dhople 1 ,N. Reiling 2 , U. Schaible 3 , F. Schmidt 1 , U. Völker 11 EMA University Greifswald, Functional Genomics, Greifswald, Germany2 Research Center Borstel, Division of Microbial Interface Biology,Borstel, Germany3 Research Center Borstel, Department of Molecular Infection Biology,Borstel, Germanylike sepsis or endocarditis [2]. Therefore, it is of interest to understand themechanism of adaptation of the pathogen upon infection as well as theresponse of its host. Proteomic studies of internalized bacteria are stronglylimited by the low number of cells recoverable from the host. With ournewly developed workflow that combines a pulse-chase SILAC approach,GFP supported enrichment of bacterial proteins by fluorescence activatedcell sorting (FACS) and gel-free mass spectrometry analysis (MS), it ispossible to monitor the proteome of S. aureus RN1HG pMV158GFPinternalized by S9 cells, human bronchial epithelial cells [3]. We identifiedabout 600 S. aureus proteins from 3-7x10 6 internalized bacteria and morethan 500 could be quantified. A further interesting host model for thisinfection assay is the A549 cell line. Those cells belong to the alveoli ofhuman lungs, produce surfactant [4] and secrete certain cytokines and havetherefore an impact also on the innate immune system. In present study weanalyzed the proteome of S. aureus after internalization by A549 cells.During a time range from 1.5-6.5 hours after infection 1-3x10 6 bacteriacould be separated from the host cells. With an optimized protocolidentification and quantification of 842 proteins could be accomplished.We could show that proteins belonging to e.g. peptidoglycan biosynthesisand glycolysis/gluconeogenesis were upregulated during infection.However, staphylococcal virulence factors which have an influence on itspathogenicity like hemolytic toxins, adhesins and enzymes which interferewith host cell signaling are mainly secreted into the host cell lumen andtherefore lost during FACS sorting. In order to make those extracellularproteins also accessible, we now established methods of enriching cellularcomponents in which S. aureus presumably resides [5]. Using densitygradient centrifugation and lipobiotin attached to magnetic beads,compartments containing S. aureus and its secreted proteins were isolatedand analyzed by LC-MS. Microscopic techniques were applied to provethe intracellular localization of S. aureus. Compartments containinginternalized S. aureus will then be isolated and lysed probably making thein vivo secretome of S. aureus accessible to proteome analysis.[1] Garzoni, C., Kelley, W.L. (2009):Trends Microbiol., 17(2), 59-65.[2] Lowy, F.D. (1998) N. Engl. J. Med., 339: 520-532.[3] Schmidt, F. et al (2010): Proteomics, 10(15): 2801-11.[4] Lieber, M. et al (1976): International Journal of Cancer, 17(1): p. 62-70.[5] Sinha, B., Fraunholz, M. (2010): International Journal of Medical Microbiology, 300(2-3):170-5.MPP061Direct activation of Legionella pneumophila glycerophospholipid:cholesterol acyltransferase PlaC by the zinc metalloproteinaseProAC. Lang* 1 , E. Rastew 2 , B. Hermes 2 , E. Siegbrecht 2 , S. Banerji 2 , A. Flieger 11 Robert Koch Institut, Division of Bacterial Infections (FG11),Wernigerode, Germany2 Robert Koch Institut, Berlin, GermanyLegionella pneumophila infects both mammalian cells and environmentalhosts, such as amoeba. Enzymes secreted by Legionella pneumophila, suchas phospholipases A (PLA) and glycerophospholipid: cholesterolacyltransferases (GCAT), may target host cell lipids and thereforecontribute to Legionnaires’ disease establishment. L. pneumophilapossesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSLfamily of lipases / acyltransferases. Enzymatic activity of these enzymesdepends on a conserved nucleophilic serine embedded into the GDSLmotif as well as on the residues aspartate and histidin together building upthe catalytic triad. The sequences of PlaA and PlaC harbour N-terminalsignal peptides for Sec and subsequent type II-dependent protein export,whereas the secretion mode of PlaD is still unclear. PlaC is the majorGCAT secreted by L. pneumophila and able to transfer free fatty acidsfrom phospholipids to cholesterol and ergosterol, additional to PLA andLPLA activities. This GCAT activity is post-transcriptionally regulated byProA, a secreted zinc metalloprotease. Since cholesterol is an importantcompound of mammalian cell membranes and ergosterol of amoebamembranes, GCAT activity might be a tool for host cell remodellingduring Legionella infection. Our aim was to characterize the mode of PlaCGCAT activation and to determine how ProA processes PlaC. Our resultsindicate that PlaC forms two protein loops due to intramolecular disulfidebonds which are both essential for PLA / GCAT activities. Analyses of thepotential cleavage site as well as loop 1 deletion mutants suggest theimportance of ProA loop deletion for GCAT activation. Our data thereforeindicate a novel enzyme inhibition / activation mechanism where loop 1displays an inhibitory effect on PlaC GCAT and full PLA activity untilPlaC is exported to the external space and subsequently activated by ProA.S. aureus was widely considered an extracellular pathogen. In the lastyears it became evident that it is able to invade and persist in nonprofessionalphagocytic cells [1]. Besides milder skin infections thisGram-positive bacterium is known to induce severe systemic infectionsBIOspektrum | Tagungsband 2012