Streptococcus pneumoniae (Spn) colonizes the upper airways of children, and the elderly, resulting in millions of deaths due to pneumonia annually.Pneumonia is an acute infection of the pulmonary parenchyma that causes cytotoxicity leading to invasion of pneumococci into the lung epithelium. Invasive bacteria produce capsule to translocate to the bloodstream causing death. All Spn strains produce abundant hydrogen peroxide (Spn-H2O2) as a byproduct of the aerobic fermentation of carbohydrates and it has a demonstrated cytotoxic activity against in vitro cultured lung epithelial cells. Despite that production of Spn-H2O2 finds optimal conditions in the lung, we lack fundamental information about the molecular mechanism driving Spn-H2O2 toxicity during replication of pneumococci in the lung parenchyma. The long-term objective of this research program is to identify the molecular mechanism(s) leading to Spn-H2O2 toxicity in the lung to find new targets for interventions. The rationale underlying this proposal is that completion will identify key molecular targets for curing pneumococcal pneumonia and preventing bacteremia. We hypothesize that oxidation of host hemoproteins by Spn-H2O2 is key in the pathophysiology of pneumococcal disease. We have made fundamental discoveries supporting this hypothesis. We demonstrated that hemoglobin induces formation of robust pneumococcal biofilms and that, concurrently, Spn-H2O2 oxidizes hemoglobin releasing heme and iron. Remarkably, the oxidation of hemoglobin by Spn-H2O2 selected for encapsulated pneumococci. The goal of this R01 proposal is therefore to characterize the role of the oxidation of hemoglobin in the pathophysiology of pneumococcal disease. Three interconnected aims are proposed to achieve our goal. In Aim 1, we will investigate the fitness cost of Spn-H2O2 oxidation of hemoglobin during biofilm formation, and the resulting selection of encapsulated pneumococci. We will study this mechanism(s) by using a series of scavengers and a systematic approach using mutants and complemented strains with different capacity to produce H2O2. Inhibition of biofilms, toxicity and the selection of encapsulated Spn will guide us towards the mechanism. In Aim 2, we will use an in vivo model of pneumococcal pneumonia, along with a quantitative confocal imaging approach, Western blot analysis, genes expression studies, and established techniques, to investigate niche-specific toxicity of Spn-H2O2, the cell's anti-oxidant response against Spn-H2O2, the oxidation of hemoglobin and the mechanism leading to selection of encapsulated pneumococci. In the final Aim 3, we will study the importance of Spn-H2O2 for the acquisition of heme/iron from hemoglobin to overcome nutrients sequestration by the host. We will address this goal by using a biochemical approach, a systematic approach with mutants and a transposon insertion sequencing screen. This innovative study will significantly advance ourknowledge about the cellular, andmolecular pathophysiology of Spn disease. This is significant because it will provide a framework for interventions aiming at oxidative reactions.

Narrative Streptococcus pneumoniae (Spn) kills millions yearly mainly due to pneumococcal pneumonia. We demonstrated that hydrogen peroxide produced by Spn strains oxidizes heme-carrying proteins, releasing and degrading heme, a molecule having a central role in the biological processes of all living organisms including humans. We will investigate the molecular and cellular mechanisms leading to the oxidation of hemoglobin by Spn and the consequences of such oxidative reactions in the pathophysiology of pneumococcal disease.