Phospholipid Sensors for Detection of Bacterial Pore-Forming Toxins
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  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.
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 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.
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