Mechanistic Investigation of the Mode of Action of the Antimicrobial Peptide Lugdunin in Lipid Membranes
by Dominik Ruppelt
Date of Examination:2023-11-13
Date of issue:2024-01-26
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Bert De Groot
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EnglishDissemination of infections originating from antibiotic-resistant bacteria constitutes an escalating threat to global healthcare systems further compounded by the stagnant development of novel antibiotics. Therefore, the discovery of antibiotic entities enacting new mechanisms of action is of paramount importance. Recently, a novel antimicrobial peptide called lugdunin was isolated from the human nasal commensal Staphylococcus lugdunensis displaying promising antimicrobial activity against a variety of antibiotic-resistant bacteria. While it is already known that lugdunin translocates protons across bacterial membranes, resulting in a dissipation of the membrane potential, its exact mechanism of action is still elusive. Understanding the mechanism of action of antimicrobial compounds is indispensable in the context of potential clinical applications. Therefore, this thesis aims at unraveling the mechanism of action of lugdunin by investigating its interaction with and activity in in vitro model membranes. Monitoring lugdunin’s tryptophan fluorescence unveiled that the peptide readily inserts into fluid lipid membranes but that its insertion and proton transport activity is hindered by the presence of components modeling Gram-negative bacterial and eukaryotic membranes. These components are known to result in membrane rigidification which could display a first approach towards a mechanism for a possible lugdunin selectivity for certain cell types. With attenuated total reflection infrared spectroscopy, lugdunin’s structure in lipid membranes was scrutinized revealing the presence of antiparallel β-sheets probably resulting from the formation of hollow tubular structures by a self-assembly process of individual lugdunin monomers. The idea of these lugdunin nanotubes acting as a potential membrane channel was strengthened by an innovative microfluidic-based proton permeation assay displaying notable similarities between lugdunin’s proton transport properties and the ones of the membrane channel gramicidin. Further electrophysiological experiments conducted with lugdunin on black lipid membranes provided crucial evidence for a lugdunin membrane channel and allowed valuable insights into the molecular architecture of lugdunin nanotubes and its conductive properties. Conclusively, this thesis presents a comprehensive model of lugdunin’s antibacterial mechanism of action comprising a rapid membrane insertion, the formation of peptide nanotubes and the subsequent translocation of ions across membranes. These results may pave the way for a lugdunin-derived antibiotic substance expanding the current antibiotic arsenal.
Keywords: Lugdunin; Model membranes; Antimicrobial peptides; Fibupeptides