Phospholipid Sensors for Detection of Bacterial Pore-Forming Toxins

Simplified phospholipid bilayer model of a cell membrane was used to accurately detect and measure activity of proteins that are secreted by bacteria pathogens.  The model consists of a molecular anchor that is chemically grafted to a solid conducting support.  Driven by the hydrophobic interaction the molecular anchor tethers phospholipid material that self-organises into an intact, low-defect density bilayer. We demonstrate that along with widely used rapid solvent exchange methodology [1] such tethered bilayers may be accomplished via vesicle fusion [2].  Also, the composition of already formed tethered bilayers may be modified by an exchange between planar bilayer and vesicles.  These methodologies allow one to vary and if necessary to adjust composition of bilayer for functional reconstitution and  detection of membrane bound proteins.

In this work we utilized tethered bilayer sensing platform to detect the activity of pore-forming toxins. Investigated toxins belong to an extensive class of cholesterol dependent cytolysins, which attach to a bilayer membrane of cells, oligomerize into a water-filled pore, and trigger cell lysis and death. The sequence of the molecular events that lead to a cell death is mediated by the cholesterol.  We demonstrate that one of such toxins, vaginolysin (VLY)  secreted by the bacteria Gardnerella vaginalis, which is a virulent factor in bacterial vaginosis, a serious gynaecological disease, that has been linked to infertility, adverse pregnancy outcomes, and increased risk for acquiring sexually transmitted diseases,  may be detected at concentrations below 0.5 nM using tethered phospholipid bilayers. These concentrations are close to physiological levels of the toxin in affected tissues. 

The detection of VLY was carried out by measuring the electrochemical impedance of a tethered bilayer membrane.  Functional reconstitution of the toxin and its oligomerization into a water-filled pore changes dramatically the electrochemical impedance spectra (EIS) of the surface-bound bilayers.  Due to proximity of a sensing bilayer to a solid surface and asymmetry of the conducting phases, the EIS cannot be modelled by simple RC parallel equivalent circuit as it is usually done in freely suspended black lipid membrane systems.  In this communication, we discuss the EIS response of tethered bilayers described earlier [3] and present several practical algorithms to evaluate the extent of membrane damage by the pore-forming toxins. The biological relevance of the EIS variation upon introduction of analyte VLY was demonstrated using VLY-neutralizing antibodies and partly inactivated mutant versions of the toxin.

The preliminary data obtained using other pore-forming cholesterol dependent toxins such as  intermedilysin and and perfringolysin is presented and compared to VLY.  In both cases the insulating properties of artificial membranes are affected by the toxins. However, to achieve maximal performance of a phospholipid sensor the composition of tethered bilayers needs to be ajdusted individually. 

References

[1] Cornell, B.; BraachMaksvytis, V.; King, L.; Osman, P.; Raguse, B.; Wieczorek, L.; Pace, R., A biosensor that uses ion-channel switches. Nature 1997,387, (6633), 580-583.

[2] Rima Budvytyte, Mindaugas Mickevicius, David J. Vanderah, Frank Heinrich, and Gintaras Valincius, Modification of Tethered Bilayers by Phospholipid Exchange with Vesicles. Langmuir. 2013 29, (13), 4320–4327.

[3] Gintaras Valincius, Tadas Meškauskas and Feliksas Ivanauskas. Electrochemical Impedance Spectroscopy of Tethered Bilayer Membranes. Langmuir. 2012, 28, (1) 977-990.