Hydrogen peroxide production by Streptococcus pneumoniae results in alpha-hemolysis by oxidation of oxy-hemoglobin to met-hemoglobin

(Spn) and other streptococci produce a greenish halo on blood agar plates referred to as α-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of α-hemolytic streptococci, including Spn, and other bacteria. The α-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply), or to a lesser extent, to lysis of erythrocytes by Spn-produced hydrogen peroxide. We investigated the molecular basis of the α-hemolysis halo produced by Spn. Wild-type strains TIGR4, D39, R6, and EF3030, and isogenic derivative Δ mutants, produced a similar α-hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout Δ/Δ mutants lacked this characteristic halo. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity, and that hydrogen peroxide contributes to the release of Ply, TIGR4 isogenic mutants Δ and Δ/Δ had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δ that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔΔ failed to produce oxidation of oxy-hemoglobin. We demonstrated that the so-called α-hemolysis halo is caused by the oxidation oxy-hemoglobin (Fe) to a non-oxygen binding met-hemoglobin (Fe) by Spn-produced hydrogen peroxide. Since Spn colonizes the human lung, oxidation of oxy-hemoglobin might have important implications for pathogenesis.