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.