title: Characterization of a Legionella pneumophila extracellular protease exhibiting hemolytic and cytotoxic activities.
abstract: A preliminary screening of selected Legionella species for proteolytic and hemolytic phenotypes suggested a correlation between these activities. To investigate the relationship of these phenotypes, a mutant strain of Legionella pneumophila deficient in the expression of a 38-kilodalton (kDa) exoprotease was isolated and characterized. This strain, designated PRT8, was also found to be nonhemolytic when screened on blood agar composed of 5% canine or guinea pig erythrocytes. Strain PRT8 was serologically and biochemically identical to the parental strain with the exception of the expression of the exoprotease. Immunoblot analysis of concentrated culture filtrates from PRT8 probed with polyclonal anti-38-kDa exoprotease serum revealed no cross-reactive peptides. To resolve the role of the exoprotease in the hemolytic phenotype, the exoprotease was purified from the culture supernatant of the parental strain by a combination of ion-exchange and hydrophobic interaction chromatography steps. The hemolytic activity was found to copurify with the proteolytic activity, and analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot revealed a single protein species exhibiting an apparent molecular mass of 38 kDa. Finally, the purified exoprotease and concentrated culture supernatant from the parental strain, but not from PRT8, exhibited cytotoxicity (minimum cytotoxic activity of 0.17 U of protease activity) in a Chinese hamster ovary cell assay. These data suggest that the exoprotease is responsible for the hemolytic and cytotoxic phenotypes described for this species and therefore may be one of several determinants associated with virulence.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
authors.forename: M G
authors.initials: MG
authors.lastname: Keen
authors.forename: P S
authors.initials: PS
authors.lastname: Hoffman
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001345:Autoradiography; D015153:Blotting, Western; D003603:Cytotoxins / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology; D006460:Hemolysin Proteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007875:Legionella / Q000201:enzymology* / Q000472:pathogenicity; D007877:Legionnaires' Disease / Q000276:immunology*; D008666:Metalloendopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D008970:Molecular Weight; D009154:Mutation
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D003603:Cytotoxins; D006460:Hemolysin Proteins; D008666:Metalloendopeptidases