Admittance change of DOPC/CHOL40% tBLMs by different variants of VLY: rVLY (black bars), rVLY mutants A162E (white bars) and A162V (grey bars).

<p>Exposure time 30 min. </p

rVLY-induced admittance change of DOPC/CHOL 40% tBLMs after 30 min preincubation with different MAbs: neutralizing MAb 9B4 (black bars), moderate neutralizing MAb 10A6 (white bars) and non-neutralizing MAb 21A5 (grey bars).

<p>Exposure of the tBLMs to rVLY time 30 min. rVLY/MAb molar ratios indicated below the abscissa.</p

Reconstitution of Cholesterol-Dependent Vaginolysin into Tethered Phospholipid Bilayers: Implications for Bioanalysis

<div><p>Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from <i>Gardnerella vaginalis</i> 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 <i>in vitro</i> 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.</p> </div

Cholesterol concentration-dependent tBLM admittance change triggered by rVLY.

<p>EIS recorded 30 min after the injection of rVLY. Incubation time 30 min. </p

Impedance Bode plots of DOPC/CHOL40% tBLMs upon exposure to rVLY solutions of different concentrations.

<p>Exposure time 30 min. (A) Impedance magnitude. (B) Impedance phase.</p

Activity of recombinant <i>G</i>. <i>vaginalis</i> sialidases in <i>E</i>. <i>coli</i> culture supernatants.

<p>The full-length <i>sld</i> gene from <i>G</i>. <i>vaginalis</i> isolates 47.3, 58.4, 60.1, 79.2, 86.1, and 114.2 was expressed in <i>E</i>. <i>coli</i>, and sialidase activity was assessed in the soluble fraction of the cell lysate. Sialidase activity was normalized to the total protein in the sample. K, soluble fraction of non-transformed <i>E</i>. <i>coli</i> cells (negative control). Error bars represent SD.</p

Phenotypic characterization of <i>Gardnerella vaginalis</i> subgroups suggests differences in their virulence potential

<div><p>The well-known genotypic and phenotypic diversity of <i>G</i>. <i>vaginalis</i> resulted in its classification into at least four subgroups (clades) with diverse genomic properties. To evaluate the virulence potential of <i>G</i>. <i>vaginalis</i> subgroups, we analyzed the virulence-related phenotypic characteristics of 14 isolates of clade 1, 12 isolates of clade 2, 8 isolates of clade 4 assessing their <i>in vitro</i> ability to grow as a biofilm, produce the toxin vaginolysin, and express sialidase activity. Significant differences in VLY production were found (<i>p</i> = 0.023), but further analysis of clade pairs did not confirm this finding. The amount of biofim did not differ significantly among the clades. Analysis of sialidase activity indicated statistically significant differences among the clades (<i>p</i> < 0.001). Production of active recombinant <i>G</i>. <i>vaginalis</i> sialidase demonstrated the link between the <i>sld</i> gene and enzymatic activity, which may be differentially regulated at the transcriptional level. Statistical classification analysis (random forests algorithm) showed that <i>G</i>. <i>vaginalis</i> clades could be best defined by the profiles of two phenotypic characteristics: sialidase activity and vaginolysin production. The results of principal component analysis and hierarchical clustering suggested that all isolates can be subgrouped into three clusters, the structures of which are determined based on phenotypic characteristics of the isolates. Clade 4 was the most homogenous group, as all isolates were found in the same cluster, which is characterized by low production of all studied virulence factors. Clade 2 isolates were mainly distributed between two clusters, whereas clade 1 isolates were found in all three clusters that were characterized by a distinct profile of phenotypic characteristics. Our findings suggest that <i>G</i>. <i>vaginalis</i> subgroups with different virulence potential might play distinct roles in vaginal microbiota.</p></div

SDS-PAGE analysis of recombinant sialidases in <i>E</i>. <i>coli</i> lysates.

<p>(A) Filtered soluble fraction of cell lysates containing full-length sialidase from <i>G</i>. <i>vaginalis</i> isolates 47.3 (lane 3), 58.4 (lane 4), 60.1 (lane 5), 79.2 (lane 6), 86.1 (lane 7), and 114.2 (lane 8). As controls, filtered soluble fractions of <i>E</i>. <i>coli</i> cell lysate before induction (lane 1) and of non-transformed cells (lane 2) were loaded on the gel. (B) The catalytic domain of sialidase from <i>G</i>. <i>vaginalis</i> isolates 86.1 and 114.2 was expressed in <i>E</i>. <i>coli</i>, and the cleared cell lysates before (lanes 1 and 3) and after (lanes 2 and 4) filtration were analyzed. M, Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific). Arrows indicate the migration positions of recombinant sialidases.</p

Results of hierarchical clustering and principal component analysis based on four characteristics: Sialidase activity, VLY production, and biofilm amount after 24 h and 48 h incubation.

<p>(A) Dendogram. The colors of the branches represent the three largest clusters. (B) PCA biplot. Axes represent the first and the second principal components (PCs). Percentages on the axes indicate the percentage of total variance explained by each PC. Arrows in the biplot represent the phenotypic characteristics. Dots indicate isolates, and the colors of the dots/isolate numbers correspond to particular clades. Closed lines indicate the areas in the space of PCs restricted by the isolates of the same clade. Shaded fields indicate areas restricted by the clusters determined in the dendogram.</p

Genotypic and phenotypic characteristics of <i>G</i>. <i>vaginalis</i> isolates.

<p>Genotypic and phenotypic characteristics of <i>G</i>. <i>vaginalis</i> isolates.</p