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

106MPV013Bartonella henselae adhesin BadA negatively regulates effectorsecretion through the VirB/D4 type IV secretion systemB. Franz* 1 , L. Yun-Yueh2 , M. Truttmann 2 , T. Riess 1 , M. Faustmann 2 ,V. Kempf 1 , C. Dehio 21 Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Institut fürMedizinische Mikrobiologie und Krankenhaushygiene, Frankfurt am Main,Germany2 Biozentrum of the University of Basel, Focal Area Infection Biology, Basel,SwitzerlandThe gram-negative, zoonotic pathogen Bartonella henselae is theaetiologic agent of cat scratch disease, bacillary angiomatosis and peliosishepatis. In recent years, two essential pathogenicity factors of B. henselaehave been investigated in detail: the trimeric autotransporter adhesinBartonella adhesin A (BadA) and the VirB/D4 type IV secretion system(T4SS). BadA mediates adherence to endothelial cells, binding tofibronectin and secretion of vascular endothelial growth factor (VEGF) inhost cells. The VirB/D4 T4SS leads to the formation of invasomes andtranslocates Bartonella effector proteins (Beps) responsible for a variety ofreactions in the host cell. Analysis of these pathogenicity factors wasperformed in two different strains of B. henselae, one expressingexclusively BadA, the other one only the VirB/D4 T4SS. Therefore, itremained unclear whether BadA and VirB/D4 T4SS functionally interactor interfere with each other.We analyzed the role of BadA and VirB/D4 T4SS when both wereexpressed in B. henselae simultaneously. Expression and function of BadAand VirB/D4 T4SS were analyzed in a variety of clinical B. henselaeisolates. However, most isolates exclusively either expressed BadA orVirB/D4 T4SS. Overexpression of full length or truncated BadA in theVirB/D4 T4SS expressing strain affected the function of the T4SSdepending on the length of BadA. In contrast, BadA dependent fibronectinbinding, VEGF secretion and adhesion to endothelial cells were notaffected by a functional VirB/D4 T4SS. Furthermore, disruption of badAin the BadA expressing strain by a transposon activated the VirB/D4T4SS. In summary, our results indicate, that BadA does not function as apartner adhesin for the VirB/D4 T4SS and, instead, BadA expressionnegatively regulates expression of the VirB/D4 T4SS by unknownmechanisms.B. Franz, L. Yun-Yueh and M. Truttmann contributed equally to this work.Also, V. Kempf and C. Dehio contributed equally.MPV014A metaproteomic analysis of a human indwelling urinarycatheter biofilm dominated by Pseudomonas aeruginosaC. Lassek* 1 , M. Burghartz 2 , D. Chaves Moreno 3 , B. Hessling 1 , A. Otto 1 ,M. Jahn 2 , D. Becher 1 , D. Pieper 3 , K. Riedel 11 Universität Greifswald, Institut für Mikrobiologie , Greifswald, Germany2 TU Braunschweig, Institut für Mikrobiologie, Braunschweig, Germany3 Helmholtz Zentrum für Infektionsforschung, Mikrobielle Interaktionenund Prozesse, Braunschweig, GermanyLong-term catheterization of the bladder leads inevitably to bacteriuria, butis mostly asymptomatic. Adaptive response of some bacteria to thecatheter environment causes an efficient biofilm formation, which cancontain 5x10^9 viable cells per centimeter. Up to now, scientistsinvestigated the microbial biofilm-forming community mainly by culturedependent methods, and only little is known about the functionaladaptation of the organisms and never a catheter-biofilm from humans wasanalyzed in depth. Our aim was to analyze a biofilm from a long-termcatheterized patient by a metaproteomic approach (1D-PAGE --> LC-ESI-MS/MS) to link structure and function of the microbial community presentin the biofilm. P.aeruginosa was found to be the predominant colonizer(160 out of 340 bacterial proteins could be assigned to P. aeruginosa), butalso other bacteria belonging to the Enterobacteriales and Bacteroidaleswere present, indicating a multispecies biofilm. The results wereconfirmed by quantitative 16S-ribosomal DNA sequencing. Abundantproteins are involved in iron and nutrient uptake, in the osmotic- and theoxidative stress response. Catheter-associated urine includes a set ofsecreted proteins which are mainly involved in iron and nutrient uptake.Additionally, the pathogens were isolated and cultured in artificial urineand in LB medium. The proteome and the secretome of P.aeruginosa wereinvestigated to elucidate the bladder specific expression and secretion ofproteins. In addition, the metaproteome contains factors of the humanimmune system, i.e. factors of the complement system and neutrophilswere found known to play an important role during host defense,indicating a symptomatic bacteriuria. Our findings help to gain a betterunderstanding of bacterial biofilms on urinary tract catheters and unravelbladder specific adaptations.MPV015Metabolic adaptations of Pseudomonas aeruginosa duringcystic fibrosis lung infectionsV. Behrends* 1 , B. Ryall 1 , J.E. Zlosnik 2 , D.A. Speert 2 , J.G. Bundy 1 , H.D. Williams 11 Imperial College, London, United Kingdom2 University of British Columbia, Vancouver, United KingdomQuestion: P. aeruginosa is a major source of nosocomial infections inimmuno-compromised patients and the leading cause of morbidity andmortality in patients with cystic fibrosis (CF). While the genetics ofadaptations to the CF lung environment during long-term infection havebeen widely studied, the physiological and metabolic impact on thebacteria is large unknown.Methods: We used untargeted metabolic profiling (metabolomics) of cellsupernatants (exometabolome analysis) to compare 179 strains,representing a series of mostly clonal lineages from 18 individual CFpatients. Isolates were collected over time periods ranging from betweenfour to twenty-four years for the individual patients.Results: We found evidence of metabolic adaptation to the CF lungenvironment: in particular, acetate production across all strains was highlysignificantly negatively associated with length of infection (P < 0.001,Spearman rank-order correlation), while uptake of metabolically‘expensive’ aromatic amino acids (Trp, Phe, Tyr) was increased. Inaddition to this parallel evolution, we observed a large degree of variationbetween the different clonal lineages.Conclusion: Our study has shown evidence of parallel metabolicadaptation of P. aeruginosa to the CF lung during chronic infection.However, isolates do not simply seem to converge on one metabolic ‘endstagephenotype’, but rather exhibit an unexpected level of metabolicdiversity between patients. Our data highlights the usefulness ofmetabolomic investigation of complex phenotypic adaptations during infection.MPV016Pneumococcal surface protein C: a multifunctionalpneumococcal virulence factor and vitronectin-binding proteinS. Voß* 1 , T. Hallström 2 , L. Petruschka 1 , K. Klingbeil 1 , K. Riesbeck 3 , P. Zipfel 2 ,S. Hammerschmidt 11 Institute for Genetics and Functional Genomics, Genetics of Microorganisms,Greifswald, Germany2 Leibniz Institute for Natural Product Research and Infection Biology, InfectionBiology, Jena, Germany3 Lund University, Laboratory Medicine, Malmö, SwedenStreptococcus pneumoniae is an asymptomatic colonizer of healthyhumans but can also cause severe local infections or even life-threateningdiseases. A prerequisite for pneumococci to colonize the upper respiratoryairways is their capability to adhere directly to host cells or indirectly byinteracting with the extracellular matrix (ECM). Pneumococcal attachmentis mediated by bacterial cell wall components and surface-exposedproteins, respectively. The major adhesin of pneumococci is thePneumococcal surface protein C (PspC) which binds to the secretorycomponent (SC) of the human polymeric Ig receptor and also recruits thecomplement regulatory protein factor H. We have also shown that hostcell-boundvitronectin (Vn), an adhesive glycoprotein present in plasmaand the ECM, is exploited by pneumococci as a molecular bridgefacilitating their adherence to and invasion into host cells by inducingproteins of the host signal transduction cascades. Although the interactionof pneumococci with vitronectin was demonstrated comprehensively, thepneumococcal adhesin for vitronectin remains unknown. Here wedemonstrate that the multifunctional PspC protein is capable to interactwith human vitronectin. Depletion of choline-binding proteins from thesurface of pneumococci resulted in decreased Vn-binding as analyzed byflow cytometry. Accordingly, PspC-deficient pneumococci showed alower capability to recruit Vn. PspC was also expressed on the surface ofnon-pathogenic Lactococcus lactis. Similar to pneumococci, theheterologous L. lactis but not the lactococcal control strain interacted withimmobilized Vn. Moreover, purified PspC protein derivativescompetitively inhibited binding of multimeric Vn to pneumococci asanalyzed by flow cytometry. Surface plasmon resonance studies wereconducted with vitronectin immobilized on a CM5 biosensor chip anddifferent PspC derivatives as analytes. PspC peptides comprising the N-terminal and helical R-domain of the native protein showed a dosedependentVn-binding. Results of a peptide SPOT array indicated that alysine-rich region as well as the SC-binding domain of PspC is probablyinvolved in binding to Vn. In conclusion, PspC exhibits vitronectinbindingactivity, and the binding site has been narrowed down to an alphahelicalregion in PspC.Bergmann S, et al. (2009) J Cell Sci 122(Pt 2):256-67.Elm C, et al. (2004) J Biol Chem 279(8): 6296-304.Hammerschmidt S. (2006) Curr Opin Microbiol 9:12-20.Hammerschmidt S, et al. (2007) J Immunol 178(9):5848-58.BIOspektrum | Tagungsband 2012