Molecular characterization of heme-carrying proteins targeted by S. pneumoniae-produced hydrogen peroxide to induce cell death

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Project summary Streptococcus pneumoniae (Spn) colonizes the lungs leading to million cases of invasive pneumococcal disease (IPD) that results in ∼1 million deaths worldwide annually. To cause IPD, pneumococcus migrates from the nasopharynx down to the lungs where it causes cytotoxicity. Spn produces several virulence factors but only a few factors, such as hydrogen peroxide (H2O2), cause cytotoxicity. Experiments using animal models of Spn disease have demonstrated that production of H2O2 plays a major role during lung colonization and for the translocation of pneumococci to the bloodstream; therefore H2O2 is essential to cause IPD. The pathophysiology of IPD includes subcellular mitochondrial damage, and apoptosis in a variety of cell types. Apoptosis in cell cultures, and in an animal model of Spn pneumonia, required of hydrogen peroxide but details of this mechanism have not been studied. In a series of breakthrough experiments we recently demonstrated that Spn-produced H2O2 oxidizes heme-carrying proteins including hemoglobin but also cytochrome C, a key molecule triggering apoptosis. We have also shown that structural changes induced by H2O2 causes the release of heme from hemoglobin and cytochrome C. Since mitochondria are essential for cell survival, and the release of cytochrome C from the mitochondria to the cytoplasm induces cell death, we hypothesize that these new discovered oxidative reaction between heme-carrying proteins and Spn-produced H2O2 is a key component of the host-cell response during the cytotoxicity observed in human lung cells and for the pathophysiology of IPD. Molecular physiological approaches, leveraged by the Molecular Center for Health and Disease (MCHD), are proposed below to assess this innovative hypothesis. In Aim 1 we will characterize the molecular and cellular mechanism(s), induced in alveolar and bronchial lung cells, by the oxidation of heme- carrying proteins. To assess this, we will investigate oxidation of mitochondrial cytochromes using available proteins and mitochondrial cytochromes purified from human immortalized and human primary differentiated cells. The specific host cell response induced by Spn-produced H2O2 will be investigated by targeted proteomics, Western blot, ELISA and FACS. Evidence from these studies will be further supported by whole transcriptome studies. Aim 2 will focus on the in-vivo consequences of hydrogen peroxide-induced oxidation of heme-carrying proteins. We will use a mouse model of pneumococcal pneumonia coupled to targeted proteomics, single-cell RNA-Seq studies along with histological evaluation and ultrastructural microscopy studies, to investigate the consequences of such oxidative reactions for healthy carriage and lung disease. The mouse model of pneumococcal disease will be utilized to evaluate the efficacy of a series of scavengers of H2O2 to decrease H2O2-associated pneumococcal carriage and/or invasive disease. This highly innovative proposal aligns with the goals of the COBRE program by integrating and leveraging Core B and Core C technologies and capabilities, mentorship, and resources provided by the MCHD.