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

lugdunensis is generally internalized into epithelial and endothelial cell linecells (5637 and EA.hy 926) and whether this internalization is alsofibronectin dependent.Methods: We characterized several clinical strains of S. lugdunensis andcompared the fibrinogen and fibronectin binding ability to the internalizationof S. lugdunensis by use of a recently described FACS-assay andtransmission electron micrography.Results: We could show for the first time that clinical isolates of S.lugdunensis that bound to fibronectin were internalized into human urinarybladder carcinoma cell line 5637 and the endothelial cell line EA.hy 926.Conclusion: The discovery of the internalization attribute of S. lugdunensisand a possible linkage to a fibronectin dependent internalization mechanismis an important step in the understanding of the pathogenicity of thispathogen.MPP019Will be presented as oral presentation with the ID MPV020!MPP020Functional properties of the putative sodium/prolinetransporter PutP of Helicobacter pyloriA. Rivera Ordaz* 1 , S. Imrich 1 , S. Rohrer 2 , R. Haas 2 , H. Jung 11 Department Biologie I, Microbiology, Biocenter, Ludwig-Maximilians-University Munich, Martinsried-Planegg, Germany2 Department Bakteriologie, Max von Pettenkofer Institute, Munich,GermanyHelicobacter pylori is a Gram-negative, pathogenic, microaerobic bacteriumcolonizing the gastric epithelium of about 50% of the world population. It isresponsible for type B gastritis, peptic ulcers, and for increasing the risk ofgastric carcinoma [1]. Successful interaction of the pathogen with its hostdoes not only require specific virulence factors, but depends also on itscapability to cope with nutrient supply and stress conditions found in thehost. Recent analyses revealed that genes encoding L-proline transport(putP) and metabolizing proteins (putA) are essential for gastric colonization[2, 3]. This research focuses on the mechanisms underlying the particularphysiological significance of L-proline and L-proline-specific systems for H.pylori and its interactions with host cells. The gene HpputP from strain P12was cloned and heterologously expressed in E. coli. HpPutP was shown tocomplement an E. coli putP mutant, thereby transport was stimulated byexternal sodium. Kinetic parameters of the sodium/proline symport processwere determined and found to be in the same order of magnitude as theEcPutP parameters. HpPutP was purified by affinity chromatography andreconstituted into proteoliposomes. Functional analyses withproteoliposomes demonstrated that the activity of HpPutP depends on anelectrochemical sodium gradient. Furthermore, sites known from EcPutP tobe of functional significance were investigated in HpPutP. By this means,residues potentially involved in sodium or proline binding and/ortranslocation were identified in HpPutP. Next, analyses of proline transportin H. pylori will be performed.[1] Suerbaum, S. and C. Josenhans (2007): Nat. Rev. Microbiol. 5, 441-452.[2] H. Kavermann, H. et al (2003): J. Exp. Med. 197, 813-822.[3] Nakajima, K.et al (2008): Biomed. Res. 29, 9-18.MPP021In vivo SigB-activity does not influence kidney geneexpression pattern in a murine Staphylococcus aureusinfection modelM. Depke* 1 , M. Burian 1 , T. Schäfer 2 , K. Ohlsen 2 , U. Völker 11 Department of Functional Genomics, Interfaculty Institute for Genetics andFunctional Genomics, Greifswald, Germany2 Institute for Molecular Infection Biology, Julius-Maximilians-University,Würzburg, GermanyRT-qPCR of mixed eukaryotic and prokaryotic RNA from infected tissueresulted in the detection of SigB-dependent higher expression levels ofasp23 and clfA in the wt strain S. aureus RN1HG, whereas, in agreementwith in vitro data, expression of hla and aur was higher in kidney tissuefrom animals infected with the sigB mutant strain.Although the virulence of sigB deficient strains is often reported to besimilar to that of wt strains, the pathomechanism of different infectionsettings might vary. Therefore, the rationale of this study was to investigatewhether the deletion of sigB would lead to a different reaction of theinfected host. Gene expression profiling indicated a highly reproducible hostkidney response to infection with S. aureus. The comparison of infectedwith non-infected samples at 120 h post infection revealed a stronginflammatory reaction. This included e. g. TLR signaling, complementsystem, antigen presentation, IFN and IL-6 signaling, but also counterregulatoryIL-10 signaling. However, the results of this study did notprovide any hints for differences in the pathomechanism of the S. aureusstrains RN1HG and ΔsigB in the selected model of i. v. infection in mice,since the host response did not differ between infections with the two strainsanalyzed. If really existing, such differences might be transient and onlyapparent at earlier time points. Effects of SigB might also be compensatedfor in in vivo infection by the interlaced pattern of other regulators. Effectsof missing activation and missing repression by SigB in the mutant mightneutralize each other.The study supports the conclusion that SigB might possess only to a lesserextent characteristics attributed to virulence factors and might act in vivomore like a virulence modulator and fine-tune bacterial reactions. SigBpossibly might be important in special niches during infection.MPP022RNase Y in the human pathogen Staphylococcus aureusG. Marincola*, C. Wolz, C. GoerkeInstitute of Microbiology and Infection Medicine (IMIT), Eberhard-Karls-University, Tübingen, GermanyBacteria are able to cope with dramatic environmental changes by rapidlyaltering gene expression. In this regulation, RNA decay, processing andmaturation play an important role. RNA decay is crucial in determiningintracellular levels of RNA species. RNA processing takes place withincomplex operons and permits the tuning of protein ratios of the cotranscribedgenes. Each of these processes requires the action ofribonucleases (RNases). The involved RNases were elucidated inEscherichia coli. In Gram-positive bacteria, RNA metabolism, so far beststudied in Bacillus subtilis, seems to be different. Sequence homologues ofsome of the E. coli enzymes that play major roles in mRNA decay, e.g.RNase E, cannot be identified in firmicutes. Recently, a new essentialendoribonuclease, RNase Y, was identified in B. subtilis. This enzyme playsa key role in mRNA turnover, in the initiation of riboswitch decay and isinvolved in the processing of the gapA operon. RNase Y is not essential inS. aureus and, therefore, we were able to construct and characterise a rnydeletion mutant. As a model for RNase Y action, we used the processing ofthe global virulence regulator saePQRS. The major transcript of the saeoperon is originated from an endonucleolytic cleavage of the primarytranscript. The rny mutant showed both defects in sae cleavage and alteredtranscription levels of the sae target gene. Moreover, other operons werealso affected by RNase Y suggesting that this enzyme is of generalimportance for mRNA processing. Microarray analysis revealed that 269transcripts were significantly upregulated and 300 were significantlydownregulated in the RNase Y mutant compare to the wild type (foldchange>2). RNase Y was previously identified as a gene affecting the virulence ofS. aureus through a silkworm infection model. Thus, the characterization ofRNAse Y mode of action and the identification of its targets is likely toenhance our understanding of the virulence of S. aureus.Staphylococcus aureus, persistent commensal of about 20% of the humanpopulation, can be transmitted to the blood after body injury or by medicaldevices like catheters. An elementary model to mimic blood stream infectionis the intra-venous infection of laboratory animals.The influence of staphylococcal i. v. infection on murine kidney geneexpression was analyzed in an in vivo model with BALB/c mice using thewild type strain RN1HG and its isogenic sigB mutant.spektrum | Tagungsband 2011