NIST-based tBLM system nears clinical application for detection of bacterial vaginosis (BV)
Utilizing NIST-developed materials that allow the formation of model tethered bilayer lipid membranes (tBLMs) at surfaces, researchers at the Institute of Biochemistry and the Department of Immunology and Cell Biology in Vilnius, Lithuania have functionally reconstituted an integral membrane protein toxin vaginolysin (VLY) and demonstrated the applicability of these tBLMs as a bioanalytical platform for the detection of bacterial vaginosis (BV). The facile detection of VLY in the tBLMs allows a more rapid (within several minutes) assessment of BV, a bacterial process linked to infertility, adverse pregnancy outcomes, post-surgery infections, and increased risks of acquiring sexually transmitted diseases. Current diagnosis of Gardnerella vaginalis, which is implicated as the virulent factor in BV, relies on time-consuming in vitro or immunoassays. As new data now demonstrates that differing VLY production levels between G. vaginalis strains may correlate with the phenotypes of BV, rapid analytical detection of VLY, as described here, could provide a new, better tool for assessing the nature and extent of this infection.
VLY is a member of a class of cholesterol-dependent cytolysins (CDCs). CDCs are produced by many gram-positive and gram-negative pathogens. CDCs bind to cholesterol-containing membranes followed by pore-formation that triggers cell lysis and death. In the current work, tBLMs were prepared with cholesterol (0 % to 40 %) and VLY reconstitution was easily detected and measured, in real-time, by electrochemical impedance spectroscopy (EIS). EIS could detect the presence of VLY as a defect or water-filled pore (hole) in the electrical insulating tBLMs down to 0.5 x 10-9 mol/L (28 ng/mL). EIS showed that the insulating properties of the tBLMs were compromised in a cholesterol-dependent fashion and neutralizing antibodies to VLY verified that the effects of VLY on the tBLMs were not due to random nonspecific interactions. In addition, the amount of VLY produced by different G. vaginalis strains and the EIS response obtained on the tBLMs were found to be well correlated.
The detailed mechanism of how VLY binds to membranes is still unknown, however, the general consensus is that cholesterol and a human receptor CD59, a glycosyl-phosphatidylinositol (GPI)–anchored membrane protein, are both involved in this interaction. While it is well-established that CD59 potentiates the hemolytic activity of VLY, the EIS data strongly suggests that VLY is capable of membrane binding and forming membrane defects in the absence of CD59.
Translation of these findings into testing of real clinical samples is slated to begin sometime early in 2014.
R. Budvytyte, M. Pleckaityte, A. Zvirbliene, D. J. Vanderah, and G. Valincius “Reconstitution of cholesterol-dependent vaginolysin into tethered phospholipid bilayers: implications for bioanalysis” PLoS One 2013 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0082536.
CONTACT: David J. Vanderah (NIST-IBBR), 240-314-6266.