The precisedescription of the interaction between peptides or proteins and lipidmembranes of cells or bacteria is rather complicated due to thecomplexity of the natural system. Therefore, various reconstitutionsystems of lipid monolayers and bilayers were developed in the lastdecades. The most simplified reconstitution system is a monomolecularlipid film (monolayer). The Langmuir-Pockels film balance can be usedto (i) prepare monolayers at the air/water interface, (ii) tocharacterize the properties of the lipid molecules in the monolayer,e.g. size per molecule and phase transitions, and (iii) to transferthe lipid monolayers under well-defined conditions onto a solidsupport using the Langmuir-Blodgett technique. Many groups uses lipidliposomes, which can be prepared in different ways resulting in smallor large liposomes which can be uni- or multi-lamellar. Liposomes arebilayer systems and thus closer to the natural membrane as themonolayers; however, it is hardly possible to reconstitute asymmetricliposomes. The reconstitution system being closest to the biologicalmembrane is that of the planar lipid bilayer prepared according tothe Montal-Mueller technique. This system has three major advantages:(i) the accessibility on both sides of the membrane, (ii) thedetermination of the formation of peptide-induced single lesions orpores can be determined, and (iii) the possibility to reconstituteasymmetric membranes. In particular, the last advantage is ofimportance for the investigation of the interactions between peptidesand the asymmetric outer membrane of Gram-negative bacteria. Thedisadvantage of this technique is the high number of experimentsneeded to achieve significant results. For some biophysicaltechniques, e.g. atomic force microscopy, it is necessary to adsorbthe mono- or bilayer on a solid support. In this case it is alwaysimportant to compare the results with those obtained from experimentsusing other reconstitution methods to avoid artifacts of the solidsupport.

We investigated the role of the composition of the lipid matrix, e.g.the presence of negatively charged lipids, of sensitive andresistance Gram-negative bacteria and of human cells for the activityof various antimicrobial peptides (AMP). The first target for AMP is,in the case of Gram-negative bacteria, the outer membrane (OM). ThisOM is an asymmetric bilayer with respect to the lipid composition.The outer leaflet consists generally of lipopolysaccharide (LPS) andthe inner leaflet of a phospholipid mixture (PL). To investigate theimportance of the lipid composition, these asymmetric membranes hasto be reconstituted as close to the natural system as possible. Wefirst performed antimicrobial tests against certain strains ofbacteria, from these bacteria we isolated the LPS, characterized itsstructure, and used it for the preparation of reconstituted lipidmono-and bilayers composed of the purified LPS.

In this contribution, mechanisms ofinteraction of peptides of the innate immune system such as defensinsand cathelicidins as well as of externally administered antibioticssuch as polymyxin B will be demonstrated.

All three membrane active peptidesinduce the permeabilization of the reconstituted membranes by theformation of lesions. We found that differences in the antimicrobialactivity of the peptides against various sensitive and resistantGram-negative bacteria can be explained solely by variations in thechemical structure of LPS. A reduction of the net negative charge ofLPS is responsible for a reduced interaction with the AMPs and thusfor resistance. Electrostatic and hydrophobic interactions are alsoimportant to explain cytotoxic effects and the activity againstGram-positive bacteria.

The results obtained by using variousbiophysical techniques and the different membrane reconstitutionsystems will be demonstrated and compared.