title: Pore-forming and haemolytic properties of the Gardnerella vaginalis cytolysin.
abstract: The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59 kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 x 10(6) HU mg-1 being obtained. The toxin-induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose-response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol-1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh-haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens thetatoxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram-positive and Gram-negative bacteria, respectively. The toxin shares several features with the family of the so-called 'sulphydryl-activated' cytolysins produced by Gram-positive bacteria, although Gvh does not truly belong to this family, being deactivated by beta-mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Cauci
authors.forename: R
authors.initials: R
authors.lastname: Monte
authors.forename: M
authors.initials: M
authors.lastname: Ropele
authors.forename: C
authors.initials: C
authors.lastname: Missero
authors.forename: T
authors.initials: T
authors.lastname: Not
authors.forename: F
authors.initials: F
authors.lastname: Quadrifoglio
authors.forename: G
authors.initials: G
authors.lastname: Menestrina
mesh_terms: D002784:Cholesterol / Q000097:blood; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003603:Cytotoxins / Q000302:isolation & purification / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects; D005260:Female; D006194:Gardnerella vaginalis / Q000472:pathogenicity / Q000502:physiology*; D006460:Hemolysin Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D006461:Hemolysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D013696:Temperature
chemical_list: D003603:Cytotoxins; D006460:Hemolysin Proteins; D008081:Liposomes; D002784:Cholesterol