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

118virulence provides novel targets for drug development. CyaB is a virulenceregulating sensor protein belonging to adenylyl cyclase (AC) Class III, aprotein family forming the second messenger cyclic adenosine 3’,5’-monophosphate (cAMP) through conserved catalytic domains, which areregulated by a diverse set of fused regulatory domains. CyaB is furtherregulated by its N-terminal MASE2 domain, which also acts as cellmembrane anchor [1].We describe here the biochemical and structural characterization of CyaB,and its inhibition by small molecules. The tertiary structure of CyaB showsthe same folding pattern, as all previously described class III ACs. CyaBindicates subtle differences in active site and inhibitor binding sites byusing the AC CyaC as a template [2].Through a genetic screen, we identified several activating mutations, thatare involved in the regulation of CyaB by the Chp virulence system, andby solving the crystal structure of the CyaB catalytic domain, we canrationalize the effects of several of these mutations and suggest that CyaBemploys regulation mechanisms similar to other Class III AC, buttriggered by other stimuli. Our results reveal mechanistic insights intophysiological and pharmacological regulation of CyaB and thus providethe basis for a better understanding of this signalling system and forexploiting it for drug development.[1] Fulcher et al, Molecular Microbiology (2010) 76(4), 889-904[2] Steegborn et al, Nature Structural & Molecular Biology (2005) 12(1), 32-37MPP038The role of 1-integrin for Yop translocation in YersiniaenterocoliticaB. Keller* 1 , E. Deuschle 1 , B. Manncke 1 , A. Siegfried 1 , R. Fässler 2 ,I.B. Autenrieth 1 , E. Bohn 11 Institute of Medical Microbiology and Hygiene, University Hospital ofTübingen, Tübingen, Germany2 Max Planck Institute of Biochemistry, Department of Molecular Medicine,Munich, GermanyYersinia enterocolitica injects effector proteins (Yops) into host cells witha Type Three Secretion System (TTSS). Injection of Yops affects severalcell functions what finally leads to immune evasion.Former studies using cultured cells showed that an interaction of theYersinia adhesion factors YadA and Invasin with 1-integrins on the hostcell site acts as a prerequisite for Yop translocation. 1-integrins aretransmembrane heterodimeric receptors which can switch between anactive and inactive conformation, and trigger various signaling cascadesinside the cell. In this study we want to show whether and how 1-integrinmediated signaling contributes to Yop translocation in vitro anddiscriminate between Inv and YadA triggered effects. For this purpose a -lactamase reporter system was used to detect and quantify Yop injection ininfected cells.We will present evidence that YadA and Invasin show striking differences howthey contribute to Yop injection.(1) Monitoring Yop translocation into epithelial and fibroblastoid cells showsthat Inv triggered Yop injection is always strictly dependent on the expressionof 1-integrins and an intact 1-cytoplasmic domain. Thereby the 1-cytoplasmic domain seems to be crucial as a binding site for the adaptor talin interms of inside-out activation and as a linker to the actin cytoskeleton. But it isnot important as a transmitter for 1-integrin mediated outside-in signaling bythe tyrosine kinases FAK, SRC or ILK. So a high affinity interaction betweenInv and 1-integrin, in which inside-out activation by talin is involved, seems tobe sufficient for Inv mediated effector translocation.(2) In contrast, only in fibroblasts but not in epithelial cells interaction of YadAwith 1-integrins is required for Yop translocation. Additionally the 1-cytoplasmic domain is only partly important for YadA triggered Yop injection.So depending on cell type 1-integrins are completely dispensible for YadAmediated Yop injection. This clearly demonstrates, that YadA can initiate Yoptranslocation also by so far unknown 1-integrin-independent mechanisms.MPP039Human formyl peptide receptor 2 senses and differentiatesenterococciD. Bloes* 1 , D. Kretschmer 1 , M. Otto 2 , A. Peschel 11 Interfaculty Insitute of Microbiology and Infection Medicine, Cellular andMolecular Microbiology, Tuebingen, Germany2 National Institute of Health, National Institute of Allergy and InfectiousDiseases, Bethesda, United StatesThe human innate immune system counteracts bacterial invaders bymultiple antimicrobial mechanisms including polymorphonuclearleukocytes (PMN), which represent the most efficient phagocytes firstoccurring at the site of infection.The human formyl peptide receptor 2 (FPR2) is a seven-transmembrane G-protein coupled receptor and is found on various cells. Recently, we couldshow that FPR2 is crucial for recruiting and activating PMN instaphylococcal infections because it senses concentrations of the majorstaphylococcal cytolysins phenol-soluble modulin (PSM) peptides [1].Moreover, FPR2 adjusts PMN responses with respect to PSM release andpathogenicity of staphylococcal species [2].Enterococci represent another group of important nosocomial pathogens.In this study, we show that not only staphylococci but also certainenterococci are capable of producing ligands for FPR2 therebyunderscoring the importance of this receptor in antibacterial host defense.PMN chemotaxis and intracellular calcium influx were induced in a dosedependentmanner by supernatants of Enterococcus faecalis andEnterococcus faecium. Only for E. faecium, this effect could be inhibitedby the S. aureus-derived FPR2-antagonist FLIPr. In agreement with this,calcium flux in receptor-transfected HL-60 cells showed that only E.faecium elicited a FPR2-specific response whereas E. faecalis did not.Also, vancomycin-resistant E. faecium isolates induced a considerablystronger response than vancomycin-susceptible isolates. However, both E.faecium and E. faecalis activated the FPR2 paralog FPR1, which sensesbacterial formylated peptides. The enterococcal genomes do not encodepeptides with apparent similarity to PSM peptides. To further characterizethe unknown FPR2 ligands produced by E. faecium, supernatants weretreated with proteases, which completely abolished the ability to stimulateFPR2 transfected HL-60 cells. This indicates that the unknown FPR2ligands of E. faecium represent peptides.In conclusion, we were able to demonstrate that certain enterococciproduce peptide-derived microbial associated molecular patterns, whichare sensed by human FPR2.[1] Kretschmer et al. (2010), Human formyl peptide receptor 2 senses highly pathogenic Staphylococcusaureus. Cell Host Microbe. Jun 25;7(6):463-73.[2] Rautenberg et al. (2011), Neutrophil responses to staphylococcal pathogens and commensals via theformyl peptide receptor 2 relates to phenol-soluble modulin release and virulence. FASEB J.Apr;25(4):1254-63.MPP040Protein-protein interaction within the Cpx-two component systemK. TschaunerUniversität Osnabrück, Molekulare Mikrobiologie, Osnabrück, GermanyTwo-component signal transduction systems (TCS) are the mainmechanisms by which bacteria sense and respond to environmental stimuli[1]. TCS typically consist of a sensor kinase (SK) and a response regulator(RR). The SK autophosphorylates upon detecting an inducing cue andtransfers the phosphoryl group to its cognate RR which now promoteschanges in cellular physiology or behavior [1]. To keep the TCS inbalance, the RR gets dephosphorylated intrinsic or due to the phosphataseactivity of the SK [1]. However, the mechanistic details about the precisesignal integration and transfer remain still unknown [2].The Cpx-envelope stress system is a well established TCS composed of themembrane-bound SK CpxA, the cytosolic RR CpxR and in addition of theaccessory protein CpxP [3]. Factors that cause cell envelope stress as e.g.pH stress, salt stress and misfolded proteins induce the Cpx-TCS [3]. Theaccessory CpxP inhibits autophosphorylation of CpxA and supports thedegradation of misfolded pilus subunits [3]. Previous functional andstructural studies suggest not only that CpxP inhibits CpxA through adirect protein-protein interaction but also indicate how CpxP act as asensor for misfolded pilus subunits, pH and salt [4]. With membrane-SPINE [5] and bacterial two-hybrid system, we were now able todemonstrate the direct physical protein-protein interaction between CpxPand CpxAin vivo. Furthermore, our data show under several Cpx-inducingconditions that CpxP is released from CpxA assigning CpxP as the sensorfor specific Cpx-inducing stimuli. Release of CpxP from CpxA is assumedto result dimerization and consequently in the autophosphorylation ofCpxA [1, 3]. Thus, our combined results lead to a deeper insight into thesignal recognition in TCS in general.A.M. Stock, V.L. Robinson and P.N. Goudreau, Annu. Rev. Biochem.69(2000), p. 183.J. Cheung and W.A. Hendrickson, Curr. Opin. Microbiol.13(2010), p. 116.S. Hunke, R. Keller and V.S. Müller, FEMS Microbiol. Lett (2011) doi: 10.1111/j.1574-6968.2011.02436.x..X. Zhou, R. Keller, R. Volkmer, N. Krauß, P. Scheerer and S. Hunke J Biol Chem286(2011), p. 9805.V.S. Müller, P.R. Jungblut, T.F. Meyer and S. Hunke, Proteomics (2011)11, p. 2124.MPP041Comparative secretome analysis of Enterococcus faecalisisolates from food and clinical originI. Hartmann* 1 , S. Giubergia 1,2 , A. Pessione 2 , E. Pessione 2 , K. Riedel 1,31 Helmholtz Centre for Infection research, Microbial Proteomics,Braunschweig, Germany2 University of Turin, Laboratorio di Biochimica e Proteomica deiMicrorganismi D.B.A.U, Turin, Italy3 Ernst-Moritz-Arndt University of Greifswald, Insitute of Microbiology,Greifswald, GermanyThe ubiquitous Gram-positive Enterococcus faecalis belongs to the groupof lactic acid bacteria and is part of the natural gut microbiota ofmammals, but is also found in a range of fermented foods, particularly inartisanal cheeses. The presence of E. faecalis in cheese can be consideredbeneficial, with its metabolic activity contributing to desired traits liketexture or flavour. Furthermore, probiotic effects have been attributed tothis organism. However, the widespread application of E. faecalis in starterBIOspektrum | Tagungsband 2012