title: Reconstitution of cholesterol-dependent vaginolysin into tethered phospholipid bilayers: implications for bioanalysis.
abstract: Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.
authors.affiliation: Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania ; Bio Complexity Department, The Niels Bohr Institute, Copenhagen University, Copenhagen, Denmark.
authors.forename: Rima
authors.initials: R
authors.lastname: Budvytyte
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Milda
authors.initials: M
authors.lastname: Pleckaityte
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.affiliation: Biomolecular Structure and Function Group, National Institute of Standards and Technology at Institute of Bioscience and Biotechnology Research, Rockville, Maryland, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vanderah
authors.affiliation: Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania.
authors.forename: Gintaras
authors.initials: G
authors.lastname: Valincius
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism; D017097:Electric Impedance; D006461:Hemolysis; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D009154:Mutation
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D008051:Lipid Bilayers; D008081:Liposomes; C542392:vaginolysin, Gardnerella vaginalis; D002784:Cholesterol