title: Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity.
abstract: The role of the single cysteine residue in the activity of the thiol-activated toxin pneumolysin was investigated using oligonucleotide-mediated, site-directed mutagenesis. Three modified toxins in which the cysteine residue was changed to an alanine, a serine, or a glycine residue were purified to homogeneity and examined for activity. The Cys-428----Ala modified toxin was indistinguishable from the wild-type recombinant toxin in terms of hemolytic activity and lytic and inhibitory effects on human polymorphonuclear leukocytes (PMN), indicating that the cysteine residue is not essential for toxin activity. The Cys-428----Ser and Cys-429----Gly modified toxins had reduced activity on erythrocytes and polymorphonuclear leukocytes, being 6 and 20 times less active than the wild type, respectively. However, all the modified toxins formed oligomers in erythrocyte membranes to the same extent as the wild-type recombinant toxin. This suggests that the cysteine residue at position 428 is involved in neither the binding of toxin to membranes nor its insertion into the membrane, and also that the formation of oligomers is not by itself sufficient for toxin activity.
authors.affiliation: Department of Microbiology, University of Leicester, United Kingdom.
authors.forename: F K
authors.initials: FK
authors.lastname: Saunders
authors.forename: T J
authors.initials: TJ
authors.lastname: Mitchell
authors.forename: J A
authors.initials: JA
authors.lastname: Walker
authors.forename: P W
authors.initials: PW
authors.lastname: Andrew
authors.forename: G J
authors.initials: GJ
authors.lastname: Boulnois
mesh_terms: D001426:Bacterial Proteins; D003167:Complement Activation; D003545:Cysteine / Q000235:genetics; D003603:Cytotoxins / Q000235:genetics / Q000633:toxicity*; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000276:immunology*; D013301:Streptolysins / Q000235:genetics / Q000633:toxicity*; D013438:Sulfhydryl Compounds
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; D013301:Streptolysins; D013438:Sulfhydryl Compounds; C032290:plY protein, Streptococcus pneumoniae; D003545:Cysteine