Functional and phylogenetic characterization of Vaginolysin, the human-specific cytolysin from Gardnerella 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 cholesterol-dependent 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.

FIG. 1.

Phylogenetic relationship between VLY and other members of the CDC family. (A) Phylogram of full-length CDC protein sequences predicted by the neighbor joining algorithm. Numbers represent calculated relative phylogenetic distances. Abbreviations for CDC proteins: LLO, listeriolysin O; IVN, ivanolysin; SLG, seeligeriolysin; SPH, sphaericolysin; ALO, anthrolysin O; CER, cereolysin; PFO, perfringolysin O; ALV, alveolysin; TET, tetanolysin O; PYO, pyolysin; MLY, mitilysin; SLO, streptolysin O; SUI, suilysin; THU, thuringensolysin. (B) Multiple alignment of undecapeptide regions from known CDCs. The predicted amino acid sequence of VLY contains a variant undecapeptide region most similar to the undecapeptide from ILY. The sequence labeled “consensus” corresponds to the undecapeptide from MLY, PLY, SUI, IVN, ALV, SPH, THU, SLO, ALO, LLO, PFO, CER, and TET. (C) Western blot of lysed G. vaginalis bacteria (GV) and purified, recombinant VLY, probed with anti-PLY monoclonal antibody.

FIG. 2.

Human-specific, cholesterol-dependent hemolytic activity of VLY. (A) Washed human (hRBC) or sheep (sRBC) erythrocytes (1% solution in PBS) were exposed to the indicated concentrations of purified recombinant VLY for 30 min, followed by pelleting of cells. Hemoglobin release was measured as optical density at 415 nm of the supernatant and normalized to 100% lysis for each species tested (P < 0.01, ANOVA). (B) Erythrocytes from various species were exposed to VLY or the non-species-specific toxin PLY (both toxins at 5 μg/ml), and lysis was measured. (C) Addition of cholesterol (Ch) at 1 μg/ml or 10 μg/ml inhibits human erythrocyte lysis by VLY (5 μg/ml) (P < 0.001, ANOVA).

FIG. 3.

Host specificity of VLY depends on the complement regulatory molecule CD59. (A) VLY-induced lysis of human erythrocytes was inhibited by monoclonal antibody to human CD59 (P < 0.0001) but not antibody to another glycosylphosphatidylinositol-anchored cell surface antigen (CD55) or mock treatment (PBS). (B) Antibody to CD59 does not inhibit PLY-mediated lysis of human erythrocytes. (C) Lactate dehydrogenase release from Chinese hamster ovary (CHO) cells transfected with empty vector (IRES) or human CD59 (IRES-hCD59) and exposed to VLY (10 μg/ml) for 30 min. Transfection of human CD59 increases VLY-mediated lysis (P < 0.0001). (D) Transfection of human CD59 into CHO cells does not affect PLY (1 μg/ml)-mediated lysis (P > 0.05).

FIG. 4.

VLY-mediated epithelial cell activation and erythrocyte lysis require P480. (A) Human cervical epithelial cell line HeLa was treated for 30 min with medium alone (−), VLY, or VLY(P480W) (1 to 10 μg/ml) prior to lysis and Western blotting with antibodies specific for total (p38) and phospho-p38 (pp38) MAPK. (B) HeLa cells were treated with VLY or VLY(P480W) (10 μg/ml) for 2 h prior to RNA extraction and assay of relative quantity of IL-8 message by real-time PCR. (C) Human (hRBC) and sheep (sRBC) erythrocytes were treated with the indicated concentrations of VLY or VLY(P480W), and hemolysis was assessed as described above. (D) Human (hRBC) erythrocytes were treated with the indicated concentrations of PLY or PLY(W435P), and hemolysis was assessed as described above.