Antibody-based detection and inhibition of vaginolysin, the Gardnerella vaginalis cytolysin

Abstract

Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae. We have recently characterized vaginolysin (VLY), the human-specific cytotoxin produced by Gardnerella vaginalis and believed to play a critical role in the pathogenesis of BV and its associated morbidities. We hypothesize that novel antibody-based strategies may be useful for detection of VLY and for inhibition of its toxic effects on human cells. Using purified toxin as an immunogen, we generated polyclonal rabbit immune serum (IS) against VLY. A western blot of G. vaginalis lysate was probed with IS and a single band (57 kD) identified. Immunofluorescence techniques using IS detected VLY production by G. vaginalis. In addition, we have developed a sandwich ELISA assay capable of VLY quantification at ng/ml concentrations in the supernatant of growing G. vaginalis. To investigate the potential inhibitory role of IS on VLY-mediated cell lysis, we exposed human erythrocytes to VLY or VLY pretreated with IS and determined the percent hemolysis. Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis. Similarly, both human cervical carcinoma cells and vaginal epithelial cells exhibited reduced cytolysis following exposure to VLY with IS compared to VLY alone. These results confirm that antibody-based techniques are an effective means of VLY detection. Furthermore, VLY antiserum functions as an inhibitor of VLY-CD59 interaction, mitigating cell lysis. These strategies may have a potential role in the diagnosis and treatment of BV.

Figure 1. Antibody techniques for the detection of VLY.

(A) Western blot of G.vaginalis 14018 lysate probed with rabbit polyclonal antiserum (1∶500,000 dilution). Numbers represent approximate MW in kD (B) Immunofluorescent detection of VLY production by G.vaginalis using pre-immune rabbit serum (bottom panel) or anti-VLY antiserum (top panel). Anti-rabbit IgG-AF488 was the secondary antibody (green). DNA staining with DAPI demonstrates bacteria in both panels (blue). Scale bar: 10 µm.

Figure 2. Quantification of VLY production in vivo.

(A) Detection of VLY in supernatants from four different strains of G. vaginalis supernatants by ELISA at various time points following inoculation of broth culture. (B) Bacterial growth (OD₆₀₀) over the course of the experiment.

Figure 3. Polyclonal immune serum inhibits VLY-mediated hemolysis.

(A) Human erythrocytes were exposed to varying concentrations of purified recombinant VLY for 30 min. Cells were pelleted, and hemoglobin release was determined by OD₄₁₅ of the supernatant. Values were normalized to 100% lysis. When indicated, VLY was preincubated with pre-bleed (VLY+PB) or immune serum (VLY+IS) diluted 1∶50 for 30 min prior to use in the assay. (B) Erythrocytes were exposed to VLY (500 ng/ml), VLY+PB, or serial dilutions of VLY+IS (P<0.01 for VLY+PB versus all VLY+IS dilutions).

Figure 4. Immune serum inhibits VLY-mediated lysis of human cervical and vaginal cells.

Human cervical (A, HeLa) or vaginal (B, VK2) epithelial cells were exposed to VLY (10 µg/ml), VLY+PB, or VLY+IS. Lysis was measured by LDH release assay following 30 min of incubation with toxin. Values were normalized to 100% lysis for each cell line (P<0.05 for VLY+IS versus VLY+PB for HeLa and VK2 cell lines).