Antibody-based detection and inhibition of vaginolysin, the Gardnerella vaginalis cytolysin.
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.
Randis
Tara M
TM
Department of Pediatrics, Columbia University, New York, NY, USA.
Kulkarni
Ritwij
R
Aguilar
Jorge L
JL
Ratner
Adam J
AJ
D000818:Animals; D000906:Antibodies; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D018957:CD59 Antigens; D016923:Cell Death / Q000276:immunology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000187:drug effects / Q000276:immunology; D005260:Female; D006194:Gardnerella vaginalis / Q000378:metabolism* / Q000472:pathogenicity; D006461:Hemolysis / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D054353:Perforin / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D002583:Uterine Cervical Neoplasms / Q000276:immunology; D014621:Vagina / Q000276:immunology / Q000382:microbiology*; D016585:Vaginosis, Bacterial / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology
D000906:Antibodies; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018957:CD59 Antigens; D007106:Immune Sera; C542392:vaginolysin, Gardnerella vaginalis; D054353:Perforin