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.

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

“…Similar EIS response was observed for other pore-forming toxins such as αHL [28,29] and vaginolysin [9]. cholesterol, already performed in literature [30].…”

“…The relevance of the importance of further studies of tBLMs is underlined by the recently reported reconstitution of membrane proteins in artificial membrane systems [49] creating an ideal platform for exploring their potential for pharmacological or biotechnological uses. Different version of lipids have been used as model building blocks to generate membranes homologue, as vesicular structures in solution (liposomes) [50], or as, tethered bilayer membrane, already tested against different toxins [9,29,51], in nanoparticle studies [52] and with soluble amyloid-beta oligomers [39]. Further improvements could be still reached related to the sensitivity of tBLMs with more detailed studies on the characteristics and properties of anchor molecules, a field continuously developing.…”

“…Usually, CDCs activity is determined by in vitro assays using red blood cells or specific cell lines [8], however recently an alternative bioanalytical technique based on the use of tethered bilayer membranes (tBLMs) was proposed [9]. The formation of defects or water-filled pores in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 3 tBLMs can be easily sensed and followed, in real-time, by electrochemical impedance spectroscopy (EIS) and opens the possibility of tBLMs use in bioanalytical applications [10,11].…”

Phospholipid Sensors for Detection of Bacterial Pore-Forming Toxins

“…In the absence of cholesterol no effect of VLY on tBLMs was detected (9). On the other hand, mutated variants of VLY or antibody treated preparations of recombinant VLY did not affect membrane integrity even in the presence of cholesterol (9).…”

Reconstitution of Functionalized Transmembrane Domains of Receptor Proteins into Biomimetic Membranes

“…12,13 Namely, tethered bilayer lipid membranes (tBLMs) are solid-supported, two-dimensional phospholipid systems which can be fabricated to closely mimic the fluid and chemically heterogeneous environment of the biological membrane, 14,15 and are well-established for investigating both peripheral and integral membrane protein interactions with a bilayer. [16][17][18][19] Delivering IMPs into the tBLMs have been performed by a number of methods, from directly synthesizing the protein in the presence of the tBLM in vitro, 9 to transferring expressed and refolded proteins from membrane-mimetic surfactants, detergents, or amphipols. 10,12,13 As a reductionist approach, synthesizing peptides comprised mainly of the TM domain of model receptor proteins offers a less complicated method for membrane deposition and biophysical characterization of integrated TM domains of model surface receptors.…”