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

(ciprofloxacin, gentamicin, sulfamethoxazole/trimethoprim, vancomycin) inlab-scale treatment plants with culture-dependent methods. Under laboratoryconditions with artificial sewage according to OECD before adding ofantibiotics only enterococci established probably due to insufficient growth,leaching from the plants, loss of cultivability of the other genera orprotozoan grazing, as described in the literature (6-8, 10). The addition ofantibiotics had no influence on the total count of bacteria and only led to adecrease in the number of enterococci by one log-level with subsequentincrease in the same order of magnitude. Without adding antibiotics therewas also an increase in the number of enterococci by one log-level, probablyby proliferation, which was shown in municipal oxidation ponds (9).Against the selected antibiotics, which were added in increasingconcentrations until 7.5 mg/L each, the enterococci developed no resistance,what was shown by comparison with isolates from a control system withoutantibiotics. However, from a concentration of 5 mg/L of each antibiotic adecrease of the nitrification rate was found.[1] Alexy & Kümmerer (2005): Korrespondenz Abwasser 52; 563-71.[2] Caballa et al (2004): Water Res 38; 2918-26.[3] Clara et al (2005): Water Res 39; 4797-807.[4] Erickson (2002): Environ Sci Technol 36; 140-45.[5] Göbel et al (2005): Environ Sci Technol 39; 3981-89.[6] Kay et al (2008): Water Res 42; 442-54.[7] Kümmerer et al (2002): BMBF-Abschlussbericht, Freiburg.[8]Menon et al (2003): Water Res 37; 4151-58.[9] Moriarty et al (2008): Appl Environ Microbiol 74; 7204-10.[10] Roszak et al (1984): Can J Microbiol 30; 34-338.[11] Thiele-Brun (2003): J Plant Nutr Soil Sci 166; 145-67.MPP015Anthrax Toxin as Trojan Horse: An N-Terminal His-TagPromotes Binding and Protective Antigen DependentUptake of C2I and Edin into CellsC. Beitzinger* 1 , M. Rolando 2 , A. Kronhardt 1 , G. Flatau 2 , M.R. Popoff 3 ,E. Lemichez 2 , R. Benz 1,41 Rudolf Virchow Center (Group Benz), Julius-Maximilians-University,Würzburg, Germany2 Batiment Archimed, Toxines microbiennes dans la relation hôtepathogènes,Nice, France3 Department of Microbiology, Institute Pastuer, Paris, France4 School of Engineering and Science, Jacobs University Bremen, GermanyBinary toxins of the AB 7-type are among the most potent and specializedbacterial protein toxins. We report that the addition of an N-terminalHis 6-tag increased the binding of LF and EF to the PA 63-channels.Interestingly, a similar effect could not be observed for the binding affinityto the C2II-channel from a His 6-tag either added to C2I nor to LF and EF.His 6-tags attached to unrelated proteins, such as the amino acids 684-1132 ofLambda phage tail protein (gpJ) or the epidermal cell differentiationinhibitor EDIN of Staphylococcus aureus promoted their binding toPA 63-channels. Here, we made the important observation that His 6-EDINexhibited voltage-dependent increase of the stability constant for binding bya factor of 25 when PA 63 and His 6-EDIN were added to the cis-side of amembrane and the trans-side was at -70 mV, which reflects the in vivosituation. Further in vivo experiments show that Anthrax- and C2-toxintranslocate into human endothelial cells. Addition of an N-terminal His 6-tagto C2I leads to its translocation into HUVECs via PA where it induces itscytotoxic effects. Finally, we demonstrated that EDIN as well as His 6-EDINenters cells through PA 63-pores but not through C2II-pores. Our resultsrevealed that a His 6-tag could induce a PA 63-dependent translocation ofproteins unrelated to the AB 7-type of toxins, possibly opening a new way toimport proteins of medical interest into cells.MPP016Cross-Reactivity of Anthrax and C2 ToxinA. Kronhardt* 1 , M. Rolando 2 , C. Beitzinger 1 , M. Leuber 1 , G. Flatau 2 ,M. Popoff 3 , E. Lemichez 2 , R. Benz 11 Rudolf Virchow Center (Group Benz), Julius-Maximilians-University,Würzburg, Germany2 Batiment Archimed, Toxines microbiennes dans la relation hôtepathogènes,Nice, France3 Department of Microbiology, Institute Pastuer, Paris, FranceBinary toxins are among the most potent bacterial protein toxins performinga cooperative mode of translocation and exhibit fatal enzymatic activities ineukaryotic cells. Anthrax and C2 toxin are the most prominent examples forthe AB 7 type of toxins. To investigate the mechanism of translocation ofthese toxins into target cells and possible cross-reactivity of toxin bindingand translocation we performed various in vitro and in vivo experiments byinterchanging the respective A and B components. Although the binding andtranslocation components Anthrax protective antigen (PA 63) and C2II of C2toxin share a sequence homology of about 35%, the results presented hereunravel biochemical and functional differences. In vitro black lipid bilayermeasurements proofed that Anthrax edema factor (EF) and lethal factor (LF)have higher affinities to bind to channels formed by C2II than C2 toxin’sC2I binds to Anthrax protective antigen (PA 63). Furthermore, we coulddemonstrate in vivo that PA in high concentration has the ability to transportthe enzymatic moiety C2I into target cells, causing actin modification andcell rounding, whereas C2II is not able to efficiently transport Anthrax EF orLF. Our findings support the commonly accepted mode of translocation ofAB 7 type of toxins. In addition, we present first-time evidence that aheterogenic combination of enzymatic and translocation components ofdifferent AB 7 toxins exhibit toxicity to primary human endothelial cells(HUVECs).MPP017Influence of different classes of gyrase inhibitors on theSOS response in Staphylococcus aureusW. Schröder*, C. Goerke, C. WolzMedical Microbiology, Eberhard-Karls-University, Tübingen, GermanyThe increase of multi-resistant bacteria is a major problem in health caresystems. Therefore, it is important to understand not only the mode of actionof antibiotics but also the mechanisms leading to the development ofresistance. Different gyrase inhibitors bind to different moieties of thegyrase which results in arrest of DNA replication. It is well documented thatthe widely used chinolone ciprofloxacin also induces the bacterial SOSresponse through activated RecA. RecA dependent cleavage of the LexArepressor results in error prone repair, which favours mutations and thereforeresistance development. A different class of gyrase inhibitors, namely theaminocoumarines bind the GyrB subunit of gyrase which leads tocompetitive inhibition of the ATPase activity of gyrase. We have analyseddifferent gyrase inhibitors and their combination with regard toantimicrobial resistance and SOS response in the human pathogenStaphylococcus aureus. Therefore, site-specific mutants (recA, lexA) wereconstructed and the influence of different gyrase inhibitors analysed bygrowth experiments, Northern Blot analysis, promotor assays and real-timeRT-PCR. RecA but not lexA mutants were shown to be more sensitivetowards several classes of antibiotics. Ciprofloxacin results in a recAdependent derepression of LexA target genes such as the error pronepolymerase SACOL1400. In contrast the aminocoumarine novobiocin leadsto transcriptional inhibition of recA as well as sarA, but induction of gyr andfnbA. These phenomenons are lexA independent and thus not related to theSOS response and probably due to other important regulators. Interestingly,the combination of ciprofloxacin and novobiocin results in abrogation of theciprofloxacin induced induction of RecA. Thus, combination of gyraseinhibitors may be beneficial to suppress the unfavourable SOS inductionaccompanied by chinolones. The mechanisms of action for novobiocin needsto be further studied, e.g. by Microarray analysis and similar drug targetscould be developed.MPP018Fibronectin binding ability of Staphylococcus lugdunensisis associated with internalization.S. Florian*, M. Lennart, S. Neumann, M. Korte, M. Kaase, S. GatermannMedical Microbiology, Ruhr-University, Bochum, GermanyQuestion: Bacterial invasion of non-phagocytic host cells is an importantpathogenicity factor for escaping the host defense system. SeveralStaphylococci, such as Staphylococcus aureus, Staphylococcussaprophyticus and Staphylococcus epidermidis, are internalized ineukaryotic cells and this mechanism is discussed as an important part of theinfection process. Fibronectin binding proteins were discussed as animportant prerequisite for internalization described for S. aureus. For S.epidermidis a fibronectin independent mechanism has been described. Twofibrinogen binding proteins were previously described for S. lugdunensis.Fibrinogen binding proteins support the adhesion of bacteria to eukaryoticcells, but were not considered as prerequisite for the internalization processfor S. aureus. To date a fibronectin binding protein for S. lugdunensis hasnot been described. We establish this study to question whether S.spektrum | Tagungsband 2011