Abstract:

Streptococcus pneumoniae (Spn) rapidly kills Staphylococcus aureus (Sau) by producing membrane-permeable hydrogen peroxide (H2O2). The mechanism by which Spn-produced H2O2 mediates Sau killing was investigated. An in vitro model that mimicked Spn-Sau contact during colonization of the nasopharynx demonstrated that Sau killing required outcompeting densities of Spn. Compared to the wildtype strain, isogenic SpnΔlctO, and SpnΔspxB, both deficient in production of H2O2, required an increased density to kill Sau. While residual H2O2 activity produced by single mutants was sufficient to eradicate Sau, a double SpnΔspxB/ΔlctO mutant was unable to kill. A collection of 20 diverse MRSA and MSSA strains showed linear sensitivity (R2=0.95) for Spn killing, but the same strains had different susceptibilities when challenged against pure H2O2 (5 mM). There was no association between Sau clonal complex and sensitivity to either Spn or H2O2. To kill Sau, Spn produced ∼180 μM of H2O2 within 4 h of incubation, while killing-defective SpnΔspxB and SpnΔspxB/lctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H2O2 when incubated with SpnΔspxB eradicated diverse Sau strains suggesting that Spn bacteria may facilitate conversion of H2O2 to a hydroxyl radical (•OH). Accordingly, Sau killing was completely blocked by incubating with scavengers of •OH radicals, Me2SO, thiourea, or sodium salicylate. The •OH was detected in the supernatant of Spn by spin trapping and electron paramagnetic resonance. Therefore, Spn produces H2O2 which is rapidly converted to a more potent oxidant, the hydroxyl radicals, to rapidly intoxicate Sau strains. Importance Streptococcus pneumoniae (Spn) strains produces hydrogen peroxide (H2O2) to kill bacteria in the upper airways, including pathogenic Staphylococcus aureus (Sau) strains. Targets of Spn-produced H2O2 have not been discovered in part because of a lack of knowledge about the underlying molecular mechanism. We demonstrated that an increased density of Spn kills Sau by means of H2O2 produced by two enzymes, SpxB and LctO. We discovered that SpxB/LctO-produced H2O2 is converted into a hydroxyl (•OH) radical that rapidly intoxicates and kills Sau. We successfully inhibited the toxicity of •OH with three different scavengers and detected •OH in the supernatant. The target(s) of the hydroxyl radicals represents a new alternative for the development of antimicrobials against Sau infections.