Pore-spanning lipid membranes as a tool to study membrane permeabilization by antimicrobial peptides
by Henrik Neubacher
Date of Examination:2017-03-09
Date of issue:2017-09-06
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Reinhard Jahn
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Abstract
English
Antimicrobial peptides (AMPs) have a high therapeutic potential in combating microbial diseases. Their modes of action are still not well understood, and screening methods to identify and improve AMPs are needed. This work explores the use of pore-spanning lipid membranes as a model system to assay AMP activity, and to investigate their modes of action. Different porous substrates are tested and evaluated for their suitability for chip-based dye translocation assays that monitor the ability of an aqueous dye to move across a lipid bilayer. Three porous substrates with different porosity, optical and surface characteristics were tested: open pore silicon nitride, closed pore silica, and closed pore anodic aluminium oxide (AAO) substrates. Lipid bilayer patches were created by spreading dye-doped giant unilamellar vesicles on the porous surface. The spreading process and membrane characteristics were characterized using confocal laser scanning microscopy in a time-resolved manner. Using the AAO support, a dye inflow assay was established. The AMPs melittin and magainin-2 were tested for their effects on lipid bilayers by monitoring dye translocation and changes in membrane fluorescence in response to administration of the respective AMP. Qualitative and quantitative measurements of the changes in membrane permeability gave insights into possible modes of action. Results suggest that melittin, but not magainin-2, acts in part by removing material from the membrane. Meanwhile, magainin-2 but not melittin is able to form pores whose size correlates with the concentration of the peptide. Finally, the assay indicated a preference of magainin-2 and δ-lysin for the liquid disordered phase of phase-separated membranes. With this, the ground work was laid for the development of an assay suitable to screen compounds for AMP activity, efficiency and efficacy, specificity and toxicity. The setup can also be used to investigate details of the modes of action of different AMPs. It has the potential for an on-chip screening platform for high throughput purposes, and applications beneficial for the in vitro research on new antimicrobial agents.
Keywords: Antimicrobial peptides; Pore-spanning lipid membranes; Anodic aluminium oxide; AAO; AMP; PSM