Streptococcus pneumoniae and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including S. pneumoniae , and other bacteria. The alpha-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 S. pneumoniae -produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by S. pneumoniae . Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δ ply mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout Δ spxB Δ lctO mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δ ply mutant strains. 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, TIGR4Δ ply and Δ spxB Δ lctO isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δ ply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4Δ spxB Δ lctO failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe 2 ) to a non-oxygen-binding met-hemoglobin (Fe 3 ) by S. pneumoniae -produced hydrogen peroxide. IMPORTANCE There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-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 S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-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 S. pneumoniae and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.
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