The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide
Infection and Immunity
The alpha-hemolysin of viridans group streptococci, which causes greening of intact erythrocytes, is a potential virulence factor as well as an important criterion for the laboratory identification of these bacteria; however, it has never been purified and characterized. The alpha-hemolysin of Streptococcus gordonii CH1 caused characteristic shifts in the A 403 , A 430 , A 578 , and A 630 of sheep hemoglobin. A spectrophotometric assay was developed and used to monitor purification of
... cation of alpha-hemolysin during extraction in organic solvents and separation by reverse-phase high-performance liquid chromatography (HPLC). The alpha-hemolysin was identical to hydrogen peroxide with respect to its effects on erythrocyte hemoglobin, oxygen-dependent synthesis by streptococci, insensitivity to proteases, inactivation by catalase, differential solubility, failure to adsorb to ion-exchange chromatography resins, and retention time on a reverse-phase HPLC column. The amount of hydrogen peroxide present in HPLC-fractionated spent culture medium was sufficient to account for all alpha-hemolytic activity observed. The viridans group of oral streptococci contains several diverse species including the following: Streptococcus pneumoniae, the principal causative agent of pneumonia and otitis media (23, 24); S. mutans and S. sobrinus, the primary etiological agents of dental caries (2, 20, 37); and S. gordonii, S. sanguis, S. oralis, and S. mitis, which frequently cause bacterial endocarditis (27, 34, 41) . The name viridans refers to the greenish discoloration (alpha-hemolysis) that these streptococci cause while growing aerobically on blood agar (29), a characteristic which has been recognized for over 90 years (28). Although alpha-hemolysin may be a significant virulence factor in the diseases caused by viridans streptococci, it is still poorly understood. This study describes the development of a fluid-phase assay for the alpha-hemolysin of S. gordonii and its purification from spent culture medium. The data indicate that the alpha-hemolysin is hydrogen peroxide on the basis of their common chemical and biological properties. The amount of hydrogen peroxide that S. gordonii produced is similar to that produced by other viridans group streptococci (10, 11, 13, 38) , indicating that it is also responsible for the alpha-hemolytic activity of these bacteria. MATERIALS AND METHODS Materials. Sheep erythrocytes, stabilized in Alsever's solution, were obtained from Crane Laboratories Inc., Syracuse, N.Y. Dehydrated brain heart infusion (BHI) and Todd-Hewitt broth (THB) media were from Difco Laboratories, Detroit, Mich., and tryptic soy broth (TSB) was from Becton Dickinson Microbiology Systems, Cockeysville, Md. Bovine liver catalase, proteinase K, pronase E, horseradish peroxidase, and 2,2Ј-azino-bis-(3-ethylbenz-thiazoline)-6-sulfonic acid (ABTS) were from Sigma Chemical Co., St. Louis, Mo. High-performance liquid chromatography (HPLC)-grade acetonitrile, 2-propanol, and water were purchased from J. T. Baker Inc., Phillipsburg, N.J. Reagent-grade HCl and 30% H 2 O 2 were from Fisher Scientific, Fairlawn, N.J.