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

项目摘要 肺炎链球菌（Spn）定植于肺部，导致数百万例侵袭性肺炎球菌感染。 疾病（IPD）每年导致全球100万人死亡。为了引起IPD，肺炎球菌从 从鼻咽到肺部引起细胞毒性。Spn产生几种毒力因子， 一些因素如过氧化氢（H2 O2）会引起细胞毒性。使用Spn动物模型的实验 已经证明，H2 O2的产生在肺定殖过程中起着重要作用， 肺炎球菌易位到血液中;因此H2 O2是导致IPD的关键。的 IPD的病理生理学包括各种细胞类型中的亚细胞线粒体损伤和细胞凋亡。 细胞培养物中的细胞凋亡，以及在Spn肺炎的动物模型中，需要过氧化氢，但细节 这一机制尚未研究。在一系列突破性的实验中，我们最近证明了 Spn产生的H2 O2氧化携带血红素的蛋白质，包括血红蛋白和细胞色素C， 触发细胞凋亡的关键分子。我们还表明，由H2 O2引起的结构变化会导致 从血红蛋白和细胞色素C中释放血红素。由于线粒体是细胞生存所必需的， 细胞色素C从线粒体释放到细胞质诱导细胞死亡，我们假设这些细胞死亡是由细胞色素C的释放引起的。 新发现的血红素携带蛋白和Spn产生的H2 O2之间的氧化反应是一个关键 在人肺细胞中观察到的细胞毒性过程中宿主细胞反应的组成部分， IPD的病理生理学分子生理学方法，由分子健康中心利用 和疾病（MCHD），下面提出评估这一创新的假设。在目标1中， 在肺泡和支气管肺细胞中诱导的血红素氧化的分子和细胞机制， 携带蛋白质。为了评估这一点，我们将研究线粒体细胞色素的氧化， 从人永生化和人初级分化的细胞中纯化的蛋白质和线粒体细胞色素 细胞将通过靶向蛋白质组学研究由Spn产生的H2 O2诱导的特异性宿主细胞应答， Western blot、ELISA和FACS。这些研究的证据将得到全转录组的进一步支持 问题研究目的2将集中在体内的后果过氧化氢诱导氧化血红素携带 proteins.我们将使用肺炎球菌肺炎的小鼠模型，结合靶向蛋白质组学，单细胞 RNA-Seq研究沿着组织学评价和超微结构显微镜研究，以研究 这种氧化反应对健康的携带和肺部疾病的后果。的小鼠模型 肺炎球菌疾病将被用来评估一系列的H2 O2清除剂，以减少 H2 O2相关肺炎球菌携带和/或侵袭性疾病。这一极具创新性的提案符合 通过集成和利用核心B和核心C技术和能力，实现COBRE计划的目标， 辅导，和MCHD提供的资源。

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.