Limits...
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.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2666159&req=5

pone-0005207-g003: 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).

Mentions: Human erythrocytes are susceptible to VLY-mediated hemolysis at low ng/ml concentrations (Figure 3A). We hypothesized that antiserum would neutralize VLY, mitigating its cytolytic activity. In order to test this hypothesis, VLY was pre-incubated with polyclonal immune serum (1∶50 dilution) for 30 minutes prior to exposure to human erythrocytes. Pre-incubation with immune serum resulted in significantly less hemolysis compared to cells exposed to untreated or pre-immune serum-treated VLY (Figure 3A). Inhibition of VLY-mediated lysis by immune serum was dose-dependent (Figure 3B) and was significant even at the highest (1∶500) dilution.


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

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

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005207-g003: 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).
Mentions: Human erythrocytes are susceptible to VLY-mediated hemolysis at low ng/ml concentrations (Figure 3A). We hypothesized that antiserum would neutralize VLY, mitigating its cytolytic activity. In order to test this hypothesis, VLY was pre-incubated with polyclonal immune serum (1∶50 dilution) for 30 minutes prior to exposure to human erythrocytes. Pre-incubation with immune serum resulted in significantly less hemolysis compared to cells exposed to untreated or pre-immune serum-treated VLY (Figure 3A). Inhibition of VLY-mediated lysis by immune serum was dose-dependent (Figure 3B) and was significant even at the highest (1∶500) dilution.

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