Functional and Phylogenetic Characterization of Vaginolysin, the Human-Specific Cytolysin fromGardnerella vaginalis

Abstract

Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus. G. vaginalis makes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate that G. vaginalis produces a toxin (vaginolysin [VLY]) that is a member of the cholesteroldependent cytolysin (CDC) family, most closely related to intermedilysin from Streptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.Bacterial vaginosis (BV) is a common and incompletely understood condition associated with adverse outcomes including preterm birth and acquisition of sexually transmitted diseases (10). Alterations of both local host immunity and the genital tract microflora appear to contribute to the pathogenesis of BV (39), and BV can be difficult to eradicate even in the setting of targeted antimicrobial therapy (4). In addition, randomized trials of antibiotics for the prevention of BV-associated preterm birth have not shown consistently beneficial effects, suggesting that host inflammatory responses set in motion early in the course of disease may contribute significantly to the consequences of infection (27).In the 1950s, Leopold (25) and then Gardner and Dukes (14) observed abundant small, pleomorphic gram-variable rods in the genital tract of women with BV. This organism, first called Haemophilus vaginalis (13) and repeatedly renamed as more information about its characteristics became available (reviewed in reference 5), is now classified as Gardnerella vaginalis, the sole member of the genus Gardnerella (16, 30). Phylogenetic analysis based on 16S rRNA places Gardnerella in the gram-positive family Bifidobacteriales. An abundance of G. vaginalis and a paucity of Lactobacillus species are characteristic of a BV-associated microflora, but the relative contribution of G. vaginalis to the pathogenesis of BV is not clear. G. vaginalis is present in essentially all cases of BV but can also be detected in a minority of asymptomatic women (1). Likewise, us...

Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes

“…The production of a cytolysin by G. vaginalis was first reported in 1990, but it has only recently been named vaginolysin, and characterized as a pore-forming cytotoxin that utilizes the complement regulatory molecule CD59 to activate the epithelial p38-mitogen-activated protein kinase pathway in human epithelial cells, leading to cell death (Gelber et al, 2008;Rottini et al, 1990). IgA antibodies against vaginolysin have been linked to the mucosal immune response during BV, further supporting the role of vaginolysin in BV pathogenesis (Cauci et al, 2002).…”

“…The biofilm incorporates other bacterial groups into its layers, suggesting that it may enable other anaerobes to colonize the vagina. G. vaginalis also produces the toxin vaginolysin, which is a member of the cholesteroldependent family of pore-forming toxins that lyses human red blood cells and vaginal epithelial cells (Gelber et al, 2008). It has been hypothesized that vaginolysin is involved in the pathogenesis of BV.…”

“…A followup study to the report that G. vaginalis in pure culture does not reliably cause BV found that fresh cultures initiated the infection more frequently (Criswell et al, 1969); this suggests that, under conditions that foster expression of virulence determinants, G. vaginalis might have greater pathogenic potential. Furthermore, recent studies have indicated that G. vaginalis is equipped with a number of virulence properties, and consequently the idea that it is the aetiological agent of BV is being revisited (Swidsinski et al, 2005;Gelber et al, 2008). Swidsinski et al (2005) have recently shown that G. vaginalis is able to form an adherent biofilm on the vaginal epithelium of women with BV.…”

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

“…ILY and the recently identified CDC from Gardnerella vaginalis, vaginolysin (VLY) do not bind directly to cholesterol but instead bind the protein receptor hCD59 (21,37). Yet the poreforming activity of both CDCs remains cholesterol dependent.…”

At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1

“…The putative cholesteroldependent lysin was found to contain the consensus undecapeptide motif for cholesterol binding but to lack the CD59 motif. Thus, although intermedilysin and vaginolysin use the human complement factor CD59 as a dependent cofactor with cholesterol to mediate binding (29), the sequence differences in the L. iners AB-1 lysin indicate that the protein does not have the specificity to bind to CD59. We noted that domain 4 of LINAB1_0216, the membrane interaction domain, was more closely related to tetanolysins than to intermedilysins (SI Appendix, Fig.…”