Identification and partial characterization of a cytolytic toxin produced by Gardnerella vaginalis

Abstract

Generation and release into the culture medium of a cytolytic toxin by Gardnerella vaginalis has been demonstrated. Addition of starch and of the nonionic detergent Tween 80 to the culture medium was essential to recover cytolytic activity. A protein with an apparent molecular mass of 61 to 63 kDa was purified from the culture supernatants showing lytic activity towards erythrocytes and nucleated cells, such as human endothelial cells and human neutrophils. The protein had marked selectivity for human erythrocytes, while erythrocytes from other species were not lysed or were lysed at much higher concentrations of the protein than those needed for human erythrocytes. The cytolytic activity was remarkably unstable in polar media, but was stabilized by nonionic detergents, by binding, or by insertion into the target cell membrane, suggesting its amphiphilic nature.

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

“…The CTox was obtained from a G. vaginalis strain, having biochemical markers identical to those of the reference strain ATCC 14018 [8,9]. The CTox was isolated from the culture supernatant and chromatographically purified in the presence of the non-ionic detergent n-/3-octylglucoside as previously described [5].…”

“…Briefly, the toxin-treated human erythrocyte membranes were prepared by incubating 3 X 108 washed erythrocytes with 20 /xg of purified CTox in a final volume of 1 ml of Krebs Ringer phosphate buffer (KRP; 120 mM NaCl, 4.9 mM KCI, 0.33 mM NaHEPO4, 16.47 mM NaEHPO4, pH 7.4), for 10 min at 4°C. After washing three times with cold 0.7 M NaCI pH 7.4 to remove the unbound toxin [5], the washed erythrocytes, resuspended in KRP, were lysed by incubating at 37°C for 20 min. Membranes were pelleted at 15000 x g for 15 min in a table top centrifuge (Beckman) and washed four times with ice-cold 5 mM phosphate buffer (pH 8.0) to remove remaining hemoglobin.…”

“…This gel allowed the discrimination of molecular masses between 40 and 450 kDa. Gels were then blotted onto cellulose nitrate and detected with a rabbit polyclonal antibody or, alternatively, with a mouse monoclonal antibody 5/1 [5]. Nitrocellulose strips were reacted with peroxidase conjugated antibodies, anti-rabbit or anti-mouse (Sigma) and probed by using 3,3'diamino-benzidine (Sigma) and H202 (Merck) as substrate.…”

“…GardnereUa vaginalis is an opportunistic pathogen harboured in the vaginal mucosa of healthy subjects [1,2], and is closely associated with bacterial vaginitis [3,4]. We have recently purified and characterized a cytolytic toxin (CTox) produced by G. vaginalis [5]. CTox is an amphiphilic protein of a molecular mass of 61 kDa.…”

“…CTox is an amphiphilic protein of a molecular mass of 61 kDa. It is specific for human cells, having a cytolytic activity on erythrocytes, polymorphonuclear leucocytes and endothelial cells, but is inactive on rabbit, guinea pig, sheep or horse erythrocytes [5]. A human erythrocyte membrane giycoprotein of 57 kDa has recently been reported to specifically bind CTox [6].…”