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

肺炎链球菌产生的过氧化氢诱导细胞死亡的血红素携带蛋白的分子特征

• 批准号: 10553870
• 负责人: Jorge Eugenio Vidal
• 金额: $ 24.62万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-06 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10553870
• 关键词: Alveolar; Alveolus; Animal Experimentation; Animal Model; Antibiotic Resistance; Antioxidants; Apoptosis; Apoptotic; BCL2 gene; Blood; Blood Circulation; Bronchi; Bronchioles; Caspase; Cell Culture Techniques; Cell Death; Cell Death Induction; Cell Differentiation process; Cell Line; Cell Survival; Cells; Centers of Research Excellence; Cessation of life; Child; Complement; Confocal Microscopy; Coupled; Cytochromes; Cytoplasm; Disease; Elderly; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial Cells; Epithelium; Evaluation; Exposure to; Family; Functional disorder; Generations; Genes; Goals; Health; Heme; Hemoglobin; Hemoglobin C; Histologic; Human; Hydrogen Peroxide; In Vitro; Induction of Apoptosis; Location; Lung; Lung diseases; Maps; Membrane Potentials; Mentorship; Metabolism; Microscopy; Mitochondria; Mitochondrial Proteins; Molecular; Nasopharynx; Necrosis; Pathway interactions; Physiological; Play; Pneumococcal Infections; Pneumococcal Pneumonia; Pneumonia; Production; Proteins; Proteomics; Reaction; Reactive Oxygen Species; Research; Research Project Grants; Resources; Role; Series; Source; Stream; Streptococcus pneumoniae; Structure of parenchyma of lung; Technology; Therapeutic; Thiourea; Vaccines; Virulence Factors; Western Blotting; cell injury; cell type; cytochrome c; cytotoxicity; efficacy evaluation; experimental study; high dimensionality; human pathogen; hydroxyurea; in vivo; innovation; lung colonization; migration; mitochondrial membrane; mouse model; mutant; oxidation; programs; response; single-cell RNA sequencing; transcriptome; transcriptomics; uptake

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.

项目概要 肺炎链球菌 (Spn) 在肺部定植，导致数百万例侵袭性肺炎球菌病例 肺炎球菌是一种每年导致全球约 100 万人死亡的疾病 (IPD)。 Spn 会从鼻咽到肺部产生细胞毒性，但仅产生多种毒力因子。 一些因素，例如过氧化氢 (H2O2)，会导致使用 Spn 动物模型的实验。 疾病已经证明，H2O2 的产生在肺部定植过程中起着重要作用，并且对于 肺炎球菌易位至血流；因此 H2O2 对于引起 IPD 至关重要。 IPD 的病理生理学包括亚细胞线粒体损伤和多种细胞类型的凋亡。 细胞培养物和 Spn 肺炎动物模型中的细胞凋亡需要过氧化氢，但详细信息 在我们最近证明的一系列突破性实验中，尚未研究过这种机制。 Spn 产生的 H2O2 氧化血红素携带蛋白，包括血红蛋白以及细胞色素 C， 我们还表明，H2O2 诱导的结构变化会导致细胞凋亡。 从血红蛋白和细胞色素 C 中释放血红素。由于线粒体对于细胞生存至关重要，并且 细胞色素 C 从线粒体释放到细胞质会诱导细胞死亡，我们发现这些 新发现的血红素携带蛋白与 Spn 产生的 H2O2 之间的氧化反应是关键 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 的病理生理学方法，由分子健康中心利用。 下面提出了评估这一创新假设的方法，我们将在目标 1 中描述这一创新假设。 通过血红素的氧化在肺泡和支气管肺细胞中诱导的分子和细胞机制 为了评估这一点，我们将使用现有的方法研究线粒体细胞色素的氧化。 从人类永生化和人类初级分化中纯化的蛋白质和线粒体细胞色素 Spn 产生的 H2O2 诱导的特定宿主细胞反应将通过靶向蛋白质组学进行研究， 这些研究的证据将得到全转录组的进一步支持。 研究目标 2 将重点关注过氧化氢诱导的血红素氧化的体内后果。 我们将使用肺炎球菌肺炎小鼠模型与靶向蛋白质组学、单细胞相结合。 RNA-Seq 研究以及组织学评估和超微结构显微镜研究，以调查 这种氧化反应对健康携带和肺部疾病的影响。 将利用肺炎球菌疾病来评估一系列 H2O2 清除剂降低肺炎球菌疾病的功效 H2O2 相关的肺炎球菌携带和/或侵袭性疾病这一高度创新的提议与 通过集成和利用核心 B 和核心 C 技术和功能来实现 COBRE 计划的目标， MCHD 提供的指导和资源。