Oxidant generation by Streptococcus pneumoniae associated with neutrophil extracellular traps

Abstract

Streptococcus pneumoniae are a common human pathogen, and the leading cause of community-acquired pneumonia world-wide. They have been shown to survive neutrophil extracellular traps (NETs), which are chromatin structures released by host neutrophils to immobilize and kill invading pathogens. It is not known how S. pneumoniae cope with the oxidants generated while they are associated with NETs. The oxidising agent hydrogen peroxide is generated by S. pneumoniae as part of their normal pyruvate metabolism, and there are a number of host enzymes in the human body that can utilize hydrogen peroxide to generate secondary oxidants. The neutrophil enzyme myeloperoxidase catalyses the formation of hypochlorous acid from hydrogen peroxide, and can be found on NETs. Given that S. pneumoniae generate hydrogen peroxide, it is possible that myeloperoxidase on NETs can utilise this hydrogen peroxide to form hypochlorous acid when the NETs come into contact with S. pneumoniae. Hypochlorous acid is bactericidal to S. pneumoniae when in media alone, though it is unclear whether they continue to generate hydrogen peroxide on NETs and subsequent hypochlorous acid is generated, or how they cope with these oxidants while associated with NETs. In this study, it was found that S. pneumoniae continue to generate hydrogen peroxide while associated with NETs at a similar rate to when in media alone, and a portion of this bacterial hydrogen peroxide was converted into hypochlorous acid by NET-associated myeloperoxidase. The amount of hypochlorous acid generated by myeloperoxidase was 2.25 nmol/107 bacteria, which was more than enough to kill the same number of bacteria in media alone. However, S. pneumoniae were not killed while associated with NETs. Exogenous hypochlorous acid was added to S. pneumoniae associated with NETs to test whether NETs actually protected the bacteria from this oxidant. It was found that S. pneumoniae associated with NETs survived concentrations of hypochlorous acid nearly 200-fold higher than when in media alone. Despite the fact that hypochlorous acid is generated when S. pneumoniae are associated with NETs, these results show that S. pneumoniae survive NET-mediated killing and remain metabolically active while associated with NETs. It is likely that the hypochlorous acid reacts with NET proteins and the other targets before ever reaching the bacteria, thus allowing S. pneumoniae to survive in the NET environment. These insights into the oxidants generated and interactions between S. pneumoniae and NETs may offer a better understanding of how pathogens persist in the lungs, and lead to further treatment options for pneumococcal diseases.