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

Abstract

Streptococcus pneumoniae (Spn) and other streptococci produce a greenish halo on blood agar plates referred to as -hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of -hemolytic streptococci, including Spn, and other bacteria. The -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 -hemolysis halo produced by Spn. Wild-type strains TIGR4, D39, R6, and EF3030, and isogenic derivative {Delta}ply mutants, produced a similar -hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout {Delta}spxB/{Delta}lctO 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 {Delta}ply and {Delta}spxB/{Delta}lctO had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4{Delta}ply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4{Delta}spxB/{Delta}lctO failed to produce oxidation of oxy-hemoglobin. We demonstrated that the so-called -hemolysis halo is caused by the oxidation oxy-hemoglobin (Fe+2) to a non-oxygen binding met-hemoglobin (Fe+3) by Spn-produced hydrogen peroxide. Since Spn colonizes the human lung, oxidation of oxy-hemoglobin might have important implications for pathogenesis. ImportanceThere is a misconception that -hemolysis observed on blood agar plates cultures of Streptococcus pneumoniae (Spn), and other -hemolytic streptococci is produced by a hemolysin, or alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type Spn strains and hemolysin (e.g., pneumolysin) knockout mutants, produced the -hemolytic halo on blood agar plates. In contrast, hydrogen peroxide defective mutants prepared in four different strains lacked the characteristic -hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of Spn and other so-called -hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the non-binding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.