Interaction of antimicrobial compounds with lipid bilayers in established and novel membrane model systems
von Johannes Schumacher
Datum der mündl. Prüfung:2020-05-06
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Michael Meinecke
EnglischInvestigating the interaction between antimicrobial compounds and membrane model systems in the form of artificial lipid bilayers provides important insights into their mode of action. The interaction of lipid side chains (LSCs) of the nucleoside antibiotic muraymycin with membranes was characterized by measuring the influx of fluorescently labeled LSCs into adhered giant unilamellar vesicles (GUVs) via confocal laser scanning microscopy (CLSM). No bilayer destabilization was detected for POPC and POPC/POPE 7:3 for any LSC at 0.8 µM. Furthermore, the partitioning of the LSCs into the vesicle bilayer was quantified. The partitioning of ω-functionalized tridecanoic acid derivates was found to correlate with the antibacterial potency of the corresponding muraymycins. Increasing PE content in the bilayer led to a decreased accumulation of LSCs. Unfunctionalized tridecanoic acid showed increased partitioning, and no negative influence of PE. This suggests that the antibacterial potency of muraymycin could be improved by exchanging the natural LSCs for long acyl chains. The mode of action of the antimicrobial cyclic peptide lugdunin was investigated with several bulk vesicle assays via fluorescence spectroscopy. The unspecific permeabilization of lipid bilayers composed of POPC and POPC/POPG 1:1 by lugdunin was shown to be negligible in a carboxyfluorescein dequenching assay. An indication for lugdunin-mediated Na+ and K+ transport was found in vesicles acidified via a potassium gradient. Follow-up experiments were conducted in vesicles containing the pH-sensitive fluorescent dye pyranine and a direct proton gradient was established via pH-change around the vesicles. Lugdunin was shown to transport protons, Na+, K+, and to a lesser degree Cs+. Variations of the assay confirmed that the rate-limiting process was peptide insertion rather than ion transport. Negative lipid headgroups had no significant effect on the insertion rate, while an influence of the peptide stock-solution solvent was observed, suggesting that a conformational change in the peptide could limit insertion. The reaction order in reference to lugdunin was found to be higher than one, indicating an oligomerization of the peptide, e.g. by stacking in the bilayer to form pores. An established membrane permeabilization assay for the characterization of antimicrobial peptides, based on pore-spanning lipid bilayers spread on optically transparent porous alumina (AAO), was further developed. The spreading of phase-separated GUVs on the porous substrate revealed lipid reorganization between solid-supported and freestanding membranes. The assay was modified to allow the application of a transmembrane potential. To this end, an electrode array in the form of silver nanowires was deposited into the porous substrate. The application of a potential to the electrode array caused a transmembrane potential across pore-spanning membranes, which was verified with the voltage-sensitive fluorescence probe DiOC2(3) via CLSM. Proton transport along the applied potential was shown by ratiometric CLSM of pyranine enclosed in the substrate pores. The interaction of pyranine with AgCl formed during the chloridation of the electrode array was found to interfere with the quantitative readout of the permeabilization rate. The orthogonal functionalization of the AAO substrate by thermal evaporation of SiO was demonstrated successfully, which reduced complexity and improved substrate stability.
Keywords: Antimicrobial peptides; Muraymycin; Lugdunin; CLSM; Fluorescence spectroscopy; Model membrane; AAO