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

OTV008Structural analysis of the polar lipids ofSphingobacterium spiritivorum and Pedobacter heparinus.B.J. Tindall* 1 , M. Nimtz 21 German Collection of Microorganisms and Cell Cultures (DSMZ),Braunschweig, Germany2 Helmholtz Center for Infection Research (HZI), Braunschweig, GermanyExamination of the polar lipids of Sphingobacterium spiritivorum andPedobacter heparinus showed that they had features typical of the aerobicbranch of the phylum Bacteroidetes, namely a single diglyceride basedphospholipid and numerous non-digylceride based lipids. Massspectrometric analysis of the isolated polar lipids of these two strainsindicated that the majority of the lipids were derived from amino acids ratherthan glycerol, to which fatty acids were linked, either by an amide linkage orby direct condensation between the fatty acid and the amino acid. Data willbe presented outlining the structures of the polar lipids in these twoorganisms.OTV009Short cationic antimicrobial peptides versus multidrugresistant bacteriaS. Ruden 1 , R. Mikut 2 , K. Hilpert* 11 Insititut of Functional Interfaces, Karlsruhe Institut of Technology (KIT),Karlsruhe, Germany2 Insititut of Applied Computer Science, KIT (Karlsruhe Institut ofTechnology (KIT), Karlsruhe, GermanyDespite decades of intensive research, antimicrobial peptides (AMPs) havenot yet revealed all their secrets; in fact, increasingly they are appearing tobe more complex than previously imagined. In recent years, it has becomeclear that they are not only able to kill Gram-positive and Gram-negativebacteria, fungi, parasites and enveloped viruses, but can also alter immuneresponse in mammals. It has been shown that short cationic AMPs can kill abroad range of multidrug resistant bacteria, indicating a different mode ofaction as the „classical antibiotics”. This feature makes them an idealcandidate for novel antimicrobial drugs that can be used to treat infectionswith multidrug resistant bacteria.Little is known about the sequence requirements of short cationic AMPs,especially for short peptides with a length between 9-13 amino acids. Withhelp of our novel technique using an artificially created luminescenceproducing Gram negative bacterium and peptide synthesis on cellulose(SPOT technology), we investigated the sequence requirements of suchpeptides. Several thousands of peptides were tested for their ability to killPseudomonas aeruginosa. Complete substitution analyses of differentindolicidin variants as well as a semi-random peptide library with about3000 members were studied. The complete substitution analysis gave usinformation about the importance of each single position whereas thepeptide library gave us broader information concerning which compositionof amino acids resulted in an active antimicrobial peptide. The data is beinganalyzed using a different quantitative structure-activity relationshipapproach (QSAR) to A) increase the percentage of active peptides in alibrary (100000 peptides were screened in silico) with very complexdescriptors and B) understand the rules by using simple descriptors thatdiscriminate between active versus inactive. For the first time, we nowunderstand the sequence requirements for short antimicrobial peptides.One critical parameter for the success of such peptides as drugs is thestability in blood serum. Here we report an easy strategy to improve the halflife time dramatically. In addition, we also added valuable information for abetter understanding of the mode of action. The results of thesemeasurements and analyses will be discussed in detail.OTV010Recombinant hydrophobin coated surfaces and theirinfluence on microbial biofilm formationA. Rieder* 1 , T. Ladnorg 1 , C. Wöll 1 , U. Obst 1 , R. Fischer 2 , T. Schwartz 11 Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT),Karlsruhe, Germany2 Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT),Karlsruhe, Germanyon a great variety of surfaces. However, the characteristics of a material andits corresponding surface properties affect the biocompatibility andconsequently bacterial adhesion and biofilm growth. In this approachrecombinant fusion hydrophobins were used for surface modification.Hydrophobins are non-toxic and non-immunogenic fungal proteins whichself-assemble on different surfaces into extremely stable monolayers in anamphiphilic manner. Recombinant hydrophobins provide the opportunity touse these surface-active proteins for large-scale surface modification ofindustrial and medical relevant materials.Thus, protocols for surface coating with recombinant fusion hydrophobinswere developed. Quartz crystal microbalance measurements were used toanalyze the adsorption behaviour of the fusion hydrophobins. Thehydrophobin coatings were characterized with water contact anglemeasurements, immunofluorescence microscopy and atomic forcemicroscopy in terms of hydrophobicity and homogeneity. The self-assemblyprocess of the recombinant fusion hydrophobins depended on the incubationtemperature and the incubation time. Fusion hydrophobins are as well suitedas natural hydrophobins for surface modification.To test the possible application of hydrophobins for antifouling coatings, thegrowth behaviour of various microorganisms was studied on hydrophobinmodified versus unmodified glass surfaces. Single bacterial strains as well asnatural bacterial communities were used to analyse biofilm formation. Apartfrom conventional plating experiments, fluorescence microscopy andmolecular-biological methods such as denaturing gradient gelelectrophoresis were applied to determine differences in the biofilm growth.The results demonstrated that the change of surface hydrophobicity and thefusion hydrophobins itself did not affect the biofilm formation.Due to their self-assembly properties, fusion hydrophobins can be used foreffective large-scale surface coating in monolayer manner. To stimulate theeffect on biofilm formation the hydrophobins can subsequently befunctionalized with already bioactive molecules like antimicrobial peptidesto influence the bacterial adhesionOTV011Investigating membrane proteins in situ by cryo-electrontomographyH. Engelhardt*, M. Eibauer, C. Hoffmann1 Molecular Structural Biology, Max Planck Institut for Biochemistry,Martinsried, GermanyCryo-electron tomography (CET) of pleomorphic microbiological objectsprovides unprecedented insight into the structural organization of nativecells and complex macromolecular assemblies [1] and opens the way toidentify and locate protein complexes and interacting macromolecules intheir natural environment [2]. However, a number of technical restrictionslimit the usable resolution to about 4 nm and impede the investigation ofmedium sized macaromolecules in the cellular context and of membraneproteins in particular.We already improved the tomographical reconstruction of membranes,demonstrating the bilayer structure of mycobacterial outer membranes inintact cells [3]. Here, we present a strategy for investigating singlemembrane proteins that are embedded in lipid bilayers. Our approachincludes improvements of the acquisition of tomographic data, the reliabledetermination and correction of the contrast transfer function of tiltprojections, the classification, alignment and the averaging of subtomogramscontaining single membrane complexes. We used the mycobacterial outermembrane protein MspA (molecular mass 160 kDa) as a test molecule,reconstituted it in lipid vesicles, and reconstructed these by CET. The 3Dmodel was considerably improved, revealed the lipid bilayer as expected,and allowed us to interprete structural details on a level of better than 1.5nm.The benefit of our approach is that it can be applied to single complexes thatare embedded in lipid vesicles as well as to (thinned) vitrified cells withoutthe necessity to artificially crystallize proteins two-dimensionally withinmembranes or to investigate the molecules in solubilized form.[1] Lucic, V. et al (2005): Annu. Ref. Biochem. 74:833-865.[2] Ortiz, J. et al (2010): J. Cell Biol. 190:613-621.[3] Hoffmann, C. et al (2008): PNAS 105:3963-3967.Biofilms represent a very successful symbiotic life form of microorganisms.They play an ambivalent role in industrial systems and can not be avoidedspektrum | Tagungsband 2011