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

114MPP020Induction of the NF-kb signal transduction pathway inresponse to Corynebacterium diphtheriae infectionL. Ott* 1 , B. Scholz 2 , K. Hasselt 3 , A. Ensser 2 , A. Burkovski 11 Lehrstuhl für Mikrobiologie, Biologie, Erlangen, Germany2 Virolgisches Institut, Erlangen, Germany3 Friedrich Bauer Institut, Bayreuth, GermanyCorynebacterium diphtheriae, the causative agent of diphtheria, has beenthoroughly studied with respect to toxin production and pili formation. Incontrast, knowledge on host responses to infection by this bacterium islimited. In this study, we analyzed epithelial cells in response tocolonization by different C. diphtheriae isolates.An NFk-B reporter cell line was used to monitor the effect of C.diphtheriae infection on human cells. Adhesion and gentamicin protectionassays revealed strains-specific differences in host pathogen interaction.Strain-specific differences and a correlation of invasion rate with inductionof NFk-B were observed in luciferase reporter gene measurements. Thiswas further supported by immune-fluorescence microscopy that showedthat translocation of p65, as a hallmark of NFk-B induction, was onlyobserved in association with cell invasion by C. diphtheriae.Our data indicate that the response of epithelial cells to C. diphtheriaeinfection is determined by the internalization of bacteria and that invasionof these cells by C. diphtheriae is an active process of these bacteria.MPP021The Na + -translocating NADH:quinone oxidoreductase (Na + -NQR) and its role in the bactericidal effect of silver ions onVibrio choleraeV. Muras* 1 , W. Steffen 1 , G. Fritz 2 , J. Steuber 11 Universität Hohenheim, Institut für Mikrobiologie, Stuttgart Hohenheim,Germany2 Uniklinik, Freiburg, GermanyThe antimicrobial effect of silver ions on a broad range of pathogenicmicroorganisms and even fungi is well known since ancient times. It is stillused today in many applications ranging from purification of waste waterto lamination of surgical instruments to control bacterial growth [1]. Thefact that there are nearly no negative effects on humans make it apromising alternative to common antibiotics. Yet the mechanism by whichAg + ions induce cell death or inhibition of growth is not fully understood.One hypothesis describing the bactericidal action of Ag + involvesinhibition of bacterial respiration [1]. A possible target molecule for Ag + isthe Na + -pumping NADH-quinone:oxidoreductase (Na + -NQR). The Na + -NQR is the main entry point for electrons into the aerobic respiratory chainof many marine and pathogenic bacteria [2]. It is a membrane-boundenzyme complex composed of six subunits (NqrABCDEF) which containsfour flavins, one 2Fe-2S cluster and ubiquinone-8 as cofactors [3]. Itsprimary function is to build up and maintain a sodium motive force (SMF)across the membrane that is used for motility and metabolic work [2]. Aninhibition by silver might therefore result in a breakdown of the SMF andthe loss of energy.Here we show that the Na + -NQR is a target for Ag + ions in Vibriocholerae. Its activity is inhibited by Ag + in the nanomolar concentrationrange both in vivo and in vitro.1. Silver, S., Bacterial silver resistance: molecular biology and uses and misuses of silver compounds. FEMSMicrobiol Rev, 2003. 27(2-3): p. 341-53.2. Duffy, E.B. and B. Barquera, Membrane topology mapping of the Na + -pumping NADH: quinoneoxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis. J Bacteriol, 2006.188(24): p. 8343-51.3. Casutt, M.S., et al., Localization and function of the membrane-bound riboflavin in the Na + -translocatingNADH:quinone oxidoreductase (Na + -NQR) from Vibrio cholerae. J Biol Chem, 2010. 285(35): p. 27088-99.MPP022The role of Yersinia enterocolitica YadA, Invasin and host cell1 integrins for Yop injection into leukocyte populations invitro and in vivoE. Deuschle* 1 , B. Keller 1 , A. Siegfried 1 , B. Manncke 1 , R. Fässler 2 ,I.B. Autenrieth 1 , E. Bohn 11 Institute for Medical Microbiology and Hygiene, Tübingen, Germany2 Max-Planck-Institut fr Biochemistry, Martinsried, GermanyDuring Yersinia infection, the bacterial type three secretion system (TTSS)is crucial for evasion of the host’s immune response. Prior to injection ofYersinia outer proteins (Yops) into the targeted cells via the TTSS,bacteria adhere to the host cells via an interaction of YadA or Invasin (Inv)with 1 integrins [1]. It was shown that 1 integrins are crucial for Yopinjection into fibroblasts [2]. Levels of Yop injection into leukocytes canbe measured by using a -lactamase reporter system for detection via flowcytometry [2]. In vitro infection of splenic leukocytes revealed that DCs,macrophages, B cells and granulocytes are infected in a similar mannerwith wildtype, Inv- or YadA-deficient strains. Experiments in a mouseinfection model revealed that Invasin plays a minor role and YadA acrucial role for Yop injection by Yersinia enterocolitica. To investigate therole of 1 integrins for Yop injection into granulocytes, B cells and T cellswe derived 1 integrin depleted splenocytes from conditional knockoutmice. Depletion of 1 integrins did not affect Yop injection mediated byYadA but reduced Yop injection triggered by Invasin, indicating that onlyInvasin triggered Yop injection is strictly 1 integrin dependent.Taken together, our data provide evidence that during systemic mouseinfection YadA but not Inv is essential for Yop injection. In consequencethis means that during mouse infection Yop injection into leukocytes canoccur also in a 1 integrin independent manner.1. Mejia, E., J.B. Bliska, and G.I. Viboud, Yersinia controls type III effector delivery into host cellsby modulating Rho activity. Plos Pathogens, 2008.4(1).2. Koberle, M., et al., Yersinia enterocolitica Targets Cells of the Innate and Adaptive ImmuneSystem by Injection of Yops in a Mouse Infection Model. Plos Pathogens, 2009.5(8).MPP023Streptococcus pneumoniae activates primary human lung cellsand stimulates exocytosis of Weibel palade bodiesS. Bergmann* 1 , M. Lüttge 2 , M. Fulde 2,3 , A. Nerlich 4 , M. Rohde 2 ,K.T. Preissner 5 , S. Hammerschmidt 6 , M. Steinert 1 , T.J. Mitchell 7 ,G.S. Chhatwal 21 Technische Universität Braunschweig, Institute for Microbiology,Braunschweig, Germany2 Helmholtz Centre for Infection Research, Braunschweig, Germany3 Hannover Medical School, Hannover, Germany4 University of Veterinary Medicine Hannover, Hannover, Germany5 Medical School, Justus-Liebig-University, Giessen, Germany6 Ernst Moritz Arndt University, Greifswald, Germany7 University of Glasgow, Glasgow, United KingdomQuestion: Streptococcus pneumoniae (pneumococcus) is a facultativepathogenic commensal colonizing the human nasopharyngeal cavity (1).Pneumococci express the pore-forming cytotoxin pneumolysin as a majorvirulence factor (2). Invasive pneumococcal infections lead toinflammatory infiltration of leukocytes into lung alveoli and to septicdissemination within the vascular system. The lung microvasculature iscovered by pulmonary endothelial cells containing special storagegranules. These granules are named Weibel-Palade bodies (WPB) andcontain the procoagulant von Willebrand factor (vWF) and IL-8, which arereleased in response to vascular injuries (3). The main question of thisstudy was focused on characterization of the interaction of pneumococciwith primary human endothelial lung cells.Methods and Results: Microscopic analyses of pneumococcal infectionwith primary human microvascular endothelial cells (HPMEC) revealed adose-dependent adherence and internalization of pneumococci.Interestingly, measurement of reactive oxygen species production usingcarboxylated H2-DCFDA indicated an activation of the cells bypneumococci. Moreover, evaluation of changes in the amount of WPBcontainingcells demonstrated a stimulation of WPB exocytosis during apneumococcal infection. The stimulation of WPB-exocytosis wasconfirmed by biochemical quantification of vWF and IL-8 secretion. Inaddition, sublytic amounts of pneumolysin stimulated vWF secretion inaddition to direct bacterial adherence. Controls of the cell morphology andevaluation of cytotoxic effects confirmed a non-altered fitness of theendothelial cells during the infection experiments.Conclusions: The release of vWF was induced after infection withpneumococci from both the apical and the basal cell surfaces, indicating astimulation of WPB exocytosis during septicemia from inside thevasculature and also following invasive pneumococcal transmigration fromthe pulmonary tissue into the bloodstream. These results demonstrate thatpneumococcal infection activates endothelial cells covering the vasculatureof humans and induces the release of pro-inflammatory and procoagulativecomponents from WPB.[1] Cartwright, K. (2002).Eur J Pediatr 161:188-95.[2] Mitchell, A. M. and Mitchell, T. J. (2010).Clin Microbiol Infect 16:411-8.[3] Rondaij, M. G., Sellink, E., Gijzen, K. A.,et al., (2004). Arterioscl Thromb Vasc Biol 24:1315-20.MPP024Msb2 shedding protects Candida albicans against antimicrobialpeptidesM. Swidergall* 1 , E. Szafranski-Schneider 1 , F. Cottier 1 , D. Tielker 1 , E. Roman 2 ,J. Pla 2 , J.F. Ernst 11 Heinrich- Heine- Universität, Molekulare Mykologie, Düsseldorf, Germany2 Universidad Complutense, Departamento de Microbiología II, Madrid, SpainMsb2 is a sensor protein in the plasma membrane of fungi. In the humanfungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinasepathway to maintain cell wall integrity and allow filamentous growth.Msb2 doubly epitope-tagged in its large extracellular and smallcytoplasmic domain was efficiently cleaved during liquid and surfacegrowth and theextracellular domain was almost quantitatively released into the growthmedium. Msb2 cleavage was independent of proteases Sap9/Sap10 andKex2. Secreted Msb2 was highly O-glycosylated by proteinmannosyltransferases including Pmt1 resulting in an apparent molecularBIOspektrum | Tagungsband 2012