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.
Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H 2 O 2 ) during growth.
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 Δply mutants, produced a similar α-hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout ΔspxB/ΔlctO 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 Δply and ΔspxB/ΔlctO had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB/ΔlctO 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.ImportanceThere 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.
Streptococcus pneumoniae (Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H2O2), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H2O2. We recently demonstrated that during infection-mimicking conditions, Spn-H2O2 oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H2O2 causes human lung cell death. Spn strains, but not H2O2-deficient SpnΔspxBΔlctO strains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cytc) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H2O2 that oxidizes hemoproteins, including Cytc, catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.
Streptococcus pneumoniae (Spn) colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite, deoxycholic acid (DoC), affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate Spn colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration DoC (0.5 mg/ml; 1.27 mM) killed all tested Spn strains (N=48) two hours post-inoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by Spn strains as soon as 10 min post-exposure. The mechanism of action did not involve activation of autolysis since the autolysis-defective double mutants Δ lytA Δ lytC and ΔspxBΔlctO were as susceptible to DoC as was the wild-type (WT). Oral streptococcal species (N=20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1x10 −9 , no spontaneous resistance was observed with DoC (srF≥1x10- 12 ). Finally, the efficacy of DoC to eradicate Spn colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with Spn EF3030 carried a median of 4.05x10 5 cfu/ml four days post-inoculation compared to 6.67x10 4 cfu/ml for mice treated with DoC. Mice in the prophylactic group had a ∼99% reduction of the pneumococcal density (median, 2.61 x10 3 cfu/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against Spn.
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