{"doi":"10.1101/2020.07.23.218966","title":"Hydrogen peroxide production by <i>Streptococcus pneumoniae</i> results in alpha-hemolysis by oxidation of oxy-hemoglobin to met-hemoglobin","abstract":"Abstract Streptococcus pneumoniae (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 ply mutants, produced a similar α-hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout Δ spxB /Δ 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 Δ ply and Δ spxB /Δ lctO had a 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. We demonstrated that the so-called α-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. Importance There is a misconception that α-hemolysis observed on blood agar plates cultures of Streptococcus pneumoniae (Spn), and other α-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 α-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide defective mutants prepared in four different strains lacked the characteristic α-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 α-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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":125185,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9608,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":570944,"name":"Faidad Khan","orcid":"0000-0002-3394-2411","position":1,"is_corresponding":false},{"id":570945,"name":"Anna Scasny","orcid":"0000-0003-3567-1157","position":2,"is_corresponding":false},{"id":571844,"name":"Zehava Eichembaun","orcid":null,"position":3,"is_corresponding":false},{"id":400645,"name":"Larry S. McDaniel","orcid":"0000-0002-1649-7563","position":4,"is_corresponding":false},{"id":449056,"name":"Jorge E. Vidal","orcid":"0000-0003-0573-5658","position":5,"is_corresponding":false},{"id":431605,"name":"Erin McDevitt","orcid":null,"position":0,"is_corresponding":true}],"reference_count":21,"raw_metadata":null,"created_at":"2026-07-18T23:15:15.482227Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}