(A) Impedance magnitude, and (B) impedance phase vs

(A) Impedance magnitude, and (B) impedance phase vs. acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal Tmem140 antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 3-Butylidenephthalide 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. == Introduction == Cholesterol-dependent cytolysins (CDCs) comprise a class of structurally related bacterial pore-forming toxins. CDCs are produced by many gram-positive pathogens [1] and have been considered as virulence factors of bacteria contributing to bacterial invasion and infection [2-4]. In addition, CDCs have been recently identified in non-pathogenic gram-negative species [5]. Vaginolysin (VLY), the toxin of CDC family, is secreted byGardnerella vaginalis[6].G. vaginalishas been identified as the prevailing inhabitant of the vaginal tract of women diagnosed with bacterial vaginosis (BV) [7,8]. BV, a disease characterized by malodorous vaginal discharge, is linked with infertility, adverse pregnancy outcomes, post-surgery infections and may increase the risk of acquiring sexually transmitted diseases [8,9]. Despite a strong correlation between the abundance ofG. vaginalisand the BV state, the role ofG. vaginaliswas considered elusive [10]. However, recent findings on a link between a structured polymicrobialG. vaginalisbiofilm covering the endometrium and fetal loss [11] is compelling evidence for the active role ofG. vaginalisin the degradation of the vaginal mucus [12] and the significance of this particular bacterium in BV. VLY is now considered as a well-recognized virulence factor ofG. vaginalis[6,13]. In addition, recent data have demonstrated that differing VLY production levels betweenG. vaginalisstrains may correlate with the phenotypes of BV [14]. Consequently, fast analytical detection of VLY and its activity, is an important issue in the assessment of the nature of the infection and could facilitate the improvement in existing methods of BV diagnosis. Commonly, CDCs activity is determined byin vitroassays using either red blood cells or cell lines such as HeLa [6,13]. Alternatively, the amount of CDCs produced by bacteria can be determined by immunoassays if the appropriate antibodies are available [13-16]. We aim at developing an alternative bioanalytical technique that would significantly simplify and speed up the measurement of the activity of the toxin 3-Butylidenephthalide so that analysis may be performed within several minutes. In our approach, we utilize the property of VLY as a member of a CDC group of toxins to bind to cholesterol-containing membranes of target cells. CDC binding leads to pore-formation that triggers cell lysis and death. The formation of defects or water-filled pores in artificial tethered bilayer membranes (tBLMs) [17,18] can be easily sensed and followed, in real-time, by electrochemical techniques, in particular, by electrochemical impedance spectroscopy (EIS) [19-21] and opens the possibility of tBLM use in bioanalytical applications. Recently, tBLMs in combination with EIS data was applied to the detection of -hemolysin (HL) in protein solutions [20] and cell cultures [22,23]. Reconstitution of HL into phospholipid bilayers has no strict requirement for cholesterol occurring via direct interaction of the protein monomer with the membrane followed by subsequent oligomerization into a heptameric pore [24]. The detailed mechanism of VLY binding is still unknown. It was shown that VLY as well as intermedilysin fromStreptococcus intermediusand lectinolysin fromStreptococcus mitisuse human CD59 as their receptor rather than cholesterol to bind to a membrane [6,25,26]. CD59 is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that 3-Butylidenephthalide blocks the formation of the complement membrane attack complex (MAC) by binding complement proteins C8 and C9 [27]. It is presumed that the requirement of CD59 in membrane binding results in the specificity.