The pore-forming toxins, inerolysin (INY) and vaginolysin (VLY), produced by vaginal bacteria Lactobacillus iners and Gardnerella vaginalis were studied using the artificial cholesterol-rich tethered bilayer membranes (tBLMs) by electrochemical techniques. The electrochemical impedance spectroscopy (EIS) of tBLMs attested for the toxin-induced impairment of the integrity of phospholipid membranes. This observation was in line with the atomic force microscopy data demonstrating formation of oligomeric protein assemblies in tBLMs. These assemblies exhibited different morphologies: VLY mostly formed complete rings, whereas INY produced arciform structures. We found that both EIS (membrane damage) and the surface plasmon resonance (protein binding) data obtained on tBLMs are in-line with the data obtained in human cell lysis experiments. EIS, however, is capable of capturing effects inaccessible for biological activity assays. Specifically, we found that the INY-induced damage of tBLMs is nearly a linear function of membrane cholesterol content, whereas VLY triggered significant damage only at high (50 mol%) cholesterol concentrations. The observed differences of INY and VLY activities on phospholipid membranes might have clinical importance: both toxin-producing bacteria have been found in healthy vagina and dysbiosis, suggesting the need for adaptation at different vaginal conditions. Our results broaden the possibilities of application of tBLMs in medical diagnostics.

Effects of amyloid beta (Aβ) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aβ oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel β sheet structural elements in HFIP oligomerized Aβ mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aβ oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.

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.

Atomic force microscopy (AFM) image analysis of supported bilayers, such as tethered bilayer membranes (tBLMs) can reveal the nature of the membrane damage by pore-forming proteins and predict the electrochemical impedance spectroscopy (EIS) response of such objects. However, automated analysis involving pore detection in such images is often non-trivial and can require AI-based object detection techniques. The specific object-detection algorithm we used to determine the defect coordinates in real AFM images was a convolutional neural network (CNN). Defect coordinates allow to predict the EIS response of tBLMs populated by the pore-forming toxins using finite element analysis (FEA) modeling. We tested if the accuracy of the CNN algorithm affected the EIS spectral features sensitive to defect densities and other physical parameters of tBLMs. We found that the EIS spectra can be predicted sufficiently well, however, systematic errors of characteristic spectral points were observed and need to be taken into account. Importantly, the comparison of predicted EIS curves with experimental ones allowed to estimate important physical parameters of tBLMs such as the specific resistance of submembrane reservoir. This reservoir separates phospholipid bilayer from the solid support. We found that the specific resistance of the reservoir amounts to $$10^{4.25 \pm 0.10}$$10 4.25 ± 0.10 $$\Omega \cdot cm$$Ω · c m which is approximately two orders of a magnitude higher compared to the specific resistance of the buffer bathing tBLMs studied in this work. We hypothesize that such effect may be related in part due to decreased concentration of ionic carriers in the submembrane due to decreased relative dielectric permittivity in this region.