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

MPP023GliT a novel thiol oxidase - implications in self resistanceand biosynthesis of gliotoxinD. Scharf* 1 , N. Remme 2 , T. Heinekamp 1 , P. Hortschansky 1 , A. Brakhage 1,3 ,C. Hertweck 2,31 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Biomolecular Chemistry, Hans Knöll Institute (HKI), Jena, Germany3 Friedrich-Schiller-University, Jena, GermanyAspergillus fumigatus and other pathogenic fungi have developed variouschemical strategies to distress, weaken or even kill their plant or animalhosts. In invasive aspergillosis, the leading cause for death inimmunocompromised patients, the fungal secondary metabolite gliotoxinplays a critical role for virulence. Gliotoxin is the prototype of a smallfamily of epipolythiodioxopiperazines (ETPs), which features uniquetransannular di- or polysulfide bridges. Extensive molecular studies haverevealed that this rare structural motif is indispensable for bioactivity and isthe key to the deleterious effects of gliotoxin.Here, we describe the function of GliT, an enzyme of the gliotoxinbiosynthesis pathway. We could reveal the activity of GliT both in vivo bymeans of feeding experiments and in vitro by heterologous overproductionand further biochemical characterisation of GliT. We proved that GliT isessential for biosynthesis of gliotoxin and therefore may play a critical rolein virulence of A. fumigatus. Furthermore, GliT confers self resistance of A.fumigatus against gliotoxin. These investigations led to the discovery of anentirely new mechanism how microorganisms could prevent self poisoningby their own toxins.[1] Scharf, D. H. et al (2010): J. Am. Chem. Soc.[2] Schrettl, M. et al (2010): PLoS Pathog.MPP024Staphylococcal serogroup L phage Φ187 useextraordinary polyglycerolphosphate wall teichoic acid asadsorption receptorV. Winstel*, G. Xia, A. PeschelInstitute of Microbiology and Infection Medicine (IMIT), MedicalMicrobiology, Eberhard-Karls-University, Tübingen, GermanyThe gram positive bacterium Staphylococcus aureus is a human pathogencausing several diseases including nosocomial infections such as sepsis andendocarditis. One very important factor for colonising, resistance toantimicrobial peptides and antibiotics is a cell wall anchored glycopolymerknown as wall teichoic acid (WTA). S. aureus strain PS 187 shows anextraordinary kind of WTA. Here the actual WTA polymer consists ofpolyglycerolphosphate modified by D-alanine (D-ala) and N-acetylgalatosamine (GalNAc). Most of the staphylococcal phages can beclassified into the serogroups A, B, D, F, G and L. Within these serogroupsonly serogroup D and L phages are able to infect S. aureus strain PS 187.Here we describe for the first time the construction of a WTA deficientmutant in this strain background via deletion of the tagO gene responsiblefor the initial step of WTA biosynthesis. Drastically reduced phosphatecontent of cell walls isolated from S. aureus PS 187 ∆tagO confirmscomplete loss of WTA. Moreover S. aureus PS 187 ∆tagO is resistant toserogroup D and serogroup L phages shown in a phage susceptibility assay.To show that phage resistance is due to the loss of WTA phage susceptibilitycould be restored by complementation using a vector expressing the tagOgene strongly suggesting that WTA is the receptor of serogroup L phages.Furthermore the question came up if loss of this special kind of WTA canlead to growth deficits. Growth kinetics show that loss ofPolyglycerolphosphate WTA is dispensable for growth in vitro.[1] Xia, G. (2010): J Biol Chem. Apr 30;285(18):13405-15.[2] Weidenmaier, C. (2004): Nat Med. Mar;10(3):243-5.[3] Peschel, A. (1999): J Biol Chem. Mar 26;274(13):8405-10.MPP025Shigella flexneri induces HIF-1alpha expression andTNF-alpha release in rat hepatocytesC.B. Cantalupo-Lima*, S.A. Santos, D.R. Andrade-JuniorInfectious and Parasitic Diseases Department, Faculty of Medicine ofUniversity of Sao Paulo, Sao Paulo - Brazil, BrazilShigella flexneri is an intracellular enteric bacteria that can disrupt gutmucosa reaching blood and liver. Hypoxia inducible factor 1alpha (HIF-1alpha) is a known transcription factor responsible for genic expressionrelated to low oxygen tensions and TNF-alpha is a key factor in the immuneresponse. In this study we investigated the ability of S. flexneri to invadecultured rat hepatocytes, and also to induce the HIF-1alpha expression andTNF-alpha release by these cells. METHODS: We cultured rat hepatocytes(Wistar) for 7 days. These cells were exposed to S. flexneri infection withmultiplicity of infection (MOI) 500:1 (5x10 8 bacteria) for 1 hour. The HIF-1alpha expression was detected by immunofluorescence and images wereanalyzed on confocal microscopy. TNF-alpha released was detected byELISA. Expression of HIF-1alpha and TNF-alpha in rat hepatocytes werecompared with groups submitted to hypoxia at incubator chamber filled withN 2 for 24 hours (final pO 2 of 40mmHg). RESULTS: The intracellularbacteria recovered reached 7x10 4 after 1 hour of infection. Infected rathepatocytes expressed HIF-1alpha mainly in the cytoplasm. The supernatantof infected hepatocytes showed higher TNF-alpha levels as compared to noninfected cells, reaching 350 pg/ml. In hypoxic groups, HIF-1alphaexpression was also observed, and TNF-alpha released decreased to 100pg/ml. CONCLUSIONS: Our results suggest that S. flexneri infection andhypoxia microenvironment were able to induce HIF-1alpha expression in rathepatocytes cultured. The increase of TNF-alpha release in infected cellswas lower in hypoxic condition. This phenomenon may be explained toprobable deficient immune response of these cells. Taken together, ourresults suggest that S. flexneri is able to invade rat hepatocytes causingfunctional alterations in these cells, specially for HIF-1alpha expression andTNF-alpha release (supported by FAPESP).MPP026Interactions of oritavancin with Lipid II and interpeptidebridge-containing Lipid II variantsT. Schneider 1 , D. Münch* 1 , A. Müller 1 , A.R. Far 2 , G. Moeck 2 , H.-G. Sahl 11 Institute of Microbiology and Biotechnology, PharmaceuticalMicrobiology, Friedrich-Wilhelms-University, Bonn, Germany2 The Medicines Company, St. Laurent, CanadaOritavancin is a semi-synthetic derivative of the glycopeptidechloroeremomycin with activity against Gram-positive pathogens, includingvancomycin-resistant staphylococci and enterococci. In contrast withvancomycin, binding of oritavancin to the cell wall precursor Lipid IIappears to involve, in addition to the D-Ala-D-Ala terminus, the interpeptidecrossbridges, as seen by nuclear magnetic resonance [1]. We studied theimpact of ORI and of its des-N-methylleucyl variant (des-ORI), which isunable to bind to the D-Ala-D-Ala terminus, on staphylococcal andenterococcal interpeptide bridge formation and Lipid IItransglycosylation/transpeptidation.[1] Kim et al (2008): J. Mol. Biol.MPP027Characterization of the major cell-associatedphospholipase A PlaB of Legionella pneumophila, the firstmember of a novel phospholipase familyK. Kuhle*, J. Bender, A. FliegerRobert Koch Institut, Wernigerode, GermanyThe lung pathogen Legionella pneumophila expresses a variety ofphospholipases potentially involved in disease-promoting processes anddevelopment of pneumonia. The recently identified major cell-associatedphospholipase A (PLA)/ lysophospholipase A (LPLA) with an additionalhemolytic activity, designed PlaB, shares no homology to previouslydescribed phospholipases. So far, it was shown that PlaB utilizes a typicaltriad of Ser-Asp-His for effective hydrolysis of phospholipids located withinthe N-terminal half of the protein for cleavage of phospholipids, such asphosphatidylglycerol (PG) and -choline (PC) as well as the respectivelysophospholipids. We further determined that PC- but not PG-hydrolyzingspektrum | Tagungsband 2011