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

Randis TM, Kulkarni R, Aguilar JL, Ratner AJ - PLoS ONE (2009)

Bottom Line: Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae.Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis.These results confirm that antibody-based techniques are an effective means of VLY detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Columbia University, New York, NY, USA.

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.

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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).
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pone-0005207-g004: 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).

Mentions: The cytolytic activity of VLY in the lower genital tract is believed to be critical for the development of bacterial vaginosis and its associated morbidities. Inhibition of VLY-mediated cell lysis in these tissues may therefore, have important physiologic relevance. Using an LDH assay, we assessed the cytolytic activity of VLY in human cervical epithelial cell (HeLa) and vaginal epithelial cells (VK2). VLY-mediated lysis of both HeLa cells (Figure 4A) and VK2 cells (Figure 4B) was markedly reduced in the setting of immune serum.


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

Randis TM, Kulkarni R, Aguilar JL, Ratner AJ - PLoS ONE (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666159&req=5

pone-0005207-g004: 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).
Mentions: The cytolytic activity of VLY in the lower genital tract is believed to be critical for the development of bacterial vaginosis and its associated morbidities. Inhibition of VLY-mediated cell lysis in these tissues may therefore, have important physiologic relevance. Using an LDH assay, we assessed the cytolytic activity of VLY in human cervical epithelial cell (HeLa) and vaginal epithelial cells (VK2). VLY-mediated lysis of both HeLa cells (Figure 4A) and VK2 cells (Figure 4B) was markedly reduced in the setting of immune serum.

Bottom Line: Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae.Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis.These results confirm that antibody-based techniques are an effective means of VLY detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Columbia University, New York, NY, USA.

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.

Show MeSH
Related in: MedlinePlus