Interaction between Streptococcus pneumoniae and Staphylococcus aureus Generates ·OH Radicals That Rapidly Kill Staphylococcus aureus Strains

Abstract

Streptococcus pneumoniae rapidly kills Staphylococcus aureus by producing membrane-permeable hydrogen peroxide (H₂O₂). The mechanism by which S. pneumoniae-produced H₂O₂ mediates S. aureus killing was investigated. An in vitro model that mimicked S. pneumoniae-S. aureus contact during colonization of the nasopharynx demonstrated that S. aureus killing required outcompeting densities of S. pneumoniae Compared to the wild-type strain, isogenic S. pneumoniae ΔlctO and S. pneumoniae ΔspxB, both deficient in production of H₂O₂, required increased density to kill S. aureus While residual H₂O₂ activity produced by single mutants was sufficient to eradicate S. aureus, an S. pneumoniae ΔspxB ΔlctO double mutant was unable to kill S. aureus A collection of 20 diverse methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains showed linear sensitivity (R² = 0.95) for S. pneumoniae killing, but the same strains had different susceptibilities when challenged with pure H₂O₂ (5 mM). There was no association between the S. aureus clonal complex and sensitivity to either S. pneumoniae or H₂O₂ To kill S. aureus, S. pneumoniae produced ∼180 μM H₂O₂ within 4 h of incubation, while the killing-defective S. pneumoniae ΔspxB and S. pneumoniae ΔspxB ΔlctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H₂O₂ incubated with S. pneumoniae ΔspxB eradicated diverse S. aureus strains, suggesting that S. pneumoniae bacteria may facilitate conversion of H₂O₂ to a hydroxyl radical (^·OH). Accordingly, S. aureus killing was completely blocked by incubation with scavengers of ^·OH radicals, dimethyl sulfoxide (Me₂SO), thiourea, or sodium salicylate. The ^·OH was detected in S. pneumoniae cells by spin trapping and electron paramagnetic resonance. Therefore, S. pneumoniae produces H₂O₂, which is rapidly converted to a more potent oxidant, hydroxyl radicals, to rapidly intoxicate S. aureus strains.IMPORTANCEStreptococcus pneumoniae strains produce hydrogen peroxide (H₂O₂) to kill bacteria in the upper airways, including pathogenic Staphylococcus aureus strains. The targets of S. pneumoniae-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 S. pneumoniae kills S. aureus by means of H₂O₂ produced by two enzymes, SpxB and LctO. We discovered that SpxB/LctO-produced H₂O₂ is converted into a hydroxyl radical (^·OH) that rapidly intoxicates and kills S. aureus 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 S. aureus infections.

Keywords: Staphylococcus aureus; Streptococcus pneumoniae; eradication; hydrogen peroxide; hydroxyl radicals.