Hydrogen peroxide is responsible for the cytotoxic effects of Streptococcus pneumoniae on primary microglia in the absence of pneumolysin

Streptococcus pneumoniae is the most common causative agent of bacterial meningitis in humans worldwide with a high morbidity and mortality rate, especially in young children and older persons. One of the best-described virulence factors of S. pneumoniae is the pore-forming toxin pneumolysin (PLY), which can harm neurons and microglia by lytic pore formation and by triggering an excessive inflammation in the brain. Another important virulence factor of S. pneumoniae is hydrogen peroxide (H₂O₂), which is mainly produced by the enzyme pyruvate oxidase (SpxB) and has also cytotoxic effects on neurons and microglia. Microglia are the resident macrophages in the brain and are responsible for the elimination of microbes and other antigens as well as for the mediation of neuroinflammation and other cellular responses.

To investigate the role of PLY and H₂O₂ during pneumococcal meningitis, we infected primary murine microglia and, for comparison, bone marrow-derived macrophages (BMDM) from mice with S. pneumoniae wild-type strain D39 as well as its isogenic mutant strains deficient for the capsule, PLY, and SpxB, respectively. After infection, we analyzed survival of the phagocytes by fluorescence microscopy, bacterial growth by quantitative plating, and phagocytosis by an antibiotic protection assay. In addition, we determined cytotoxic effects of H₂O₂ and recombinant PLY by fluorescence microscopy or lactate dehydrogenase assay, respectively.

We found that microglia were killed during pneumococcal infection even in the absence of PLY, whereas BMDM survived. Treatment with recombinant PLY showed that very high concentrations were needed to harm the phagocytes but these concentrations were not reached during in vitro pneumococcal infection. In contrast, exogenously added catalase abolished the cytotoxic effects of H₂O₂ towards microglia and BMDM, and infection with pneumococci deficient for SpxB resulted in reduced killing of microglia.

To conclude, H₂O₂ produced by pneumococci was able to cause microglial cell death in the absence of PLY, whereas PLY was not released in sufficient concentrations to damage microglia under these experimental conditions.