The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

From LUVs to GUVs─How to Cover Micrometer-Sized Pores with Membranes.

Pore-spanning membranes (PSMs) are a versatile tool to investigate membrane-confined processes in a bottom-up approach. Pore sizes in the micrometer range are most suited to visualize PSMs using fluorescence microscopy. However, the preparation of these PSMs relies on the spreading of giant unilamellar vesicles (GUVs). GUV production faces several limitations. Thus, alternative ways to generate PSMs starting from large or small unilamellar vesicles that are more reproducibly prepared are highly desirable. Here we describe a method to produce PSMs obtained from large unilamellar vesicles, making use of droplet-stabilized GUVs generated in a microfluidic device. We analyzed the lipid diffusion in the free-standing and supported parts of the PSMs using z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching experiments in combination with finite element simulations. Employing atomic force indentation experiments, we also investigated the mechanical properties of the PSMs. Both lipid diffusion constants and lateral membrane tension were compared to those obtained on PSMs derived from electroformed GUVs, which are known to be solvent- and detergent-free, under otherwise identical conditions. Our results demonstrate that the lipid diffusion, as well as the mechanical properties of the resulting PSMs, is almost unaffected by the GUV formation procedure but depends on the chosen substrate functionalization. With the new method in hand, we were able to reconstitute the syntaxin-1A transmembrane domain in microfluidic GUVs and PSMs, which was visualized by fluorescence microscopy.