title: Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.
abstract: We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.
authors.affiliation: Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania.
authors.forename: Giulio
authors.initials: G
authors.lastname: Preta
authors.affiliation: Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania.
authors.forename: Marija
authors.initials: M
authors.lastname: Jankunec
authors.affiliation: NIST Center for Neutron Research, Gaithersburg, MD 20899, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Heinrich
authors.affiliation: Institute of Life Science, Swansea University Medical School, Swansea SA2 8PP, United Kingdom.
authors.forename: Sholeem
authors.initials: S
authors.lastname: Griffin
authors.affiliation: Institute of Life Science, Swansea University Medical School, Swansea SA2 8PP, United Kingdom.
authors.forename: Iain Martin
authors.initials: IM
authors.lastname: Sheldon
authors.affiliation: Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania. Electronic address: gintaras.valincius@bchi.vu.lt.
authors.forename: Gintaras
authors.initials: G
authors.lastname: Valincius
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D058266:Dielectric Spectroscopy; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D006835:Hydrazones / Q000737:chemistry / Q000494:pharmacology; D008051:Lipid Bilayers / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009154:Mutation
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D006835:Hydrazones; D008051:Lipid Bilayers; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; C108448:Pyolysin, Arcanobacterium pyogenes
keywords: Cholesterol-dependent cytolysins; Dynasore; Electrochemical impedance spectroscopy; Tethered bilayer membranes