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.

项目摘要 肺炎链球菌（SPN）在肺部定居，导致数百万例侵入性肺炎病例 疾病（IPD）每年在全球造成约100万人死亡。为了引起IPD，肺炎球菌从 鼻咽降低到引起细胞毒性的肺部。 SPN产生多个病毒因素，但仅 几种因素，例如过氧化氢（H2O2）引起细胞毒性。使用SPN动物模型的实验 疾病已经表明，H2O2的产生在肺定植期间起着重要作用 肺炎球菌转移到血液中；因此，H2O2对于引起IPD至关重要。这 IPD的病理生理包括各种细胞类型的细胞细胞软件损伤和凋亡。 细胞培养物中的凋亡，以及SPN肺炎的动物模型，是过氧化氢所必需的，但详细介绍了 这种机制尚未研究。在一系列突破实验中，我们最近证明了 SPN生产的H2O2氧化了血红素携带的蛋白质，包括血红蛋白，但也包括细胞色素C，A 关键分子触发凋亡。我们还表明，H2O2诱导的结构变化导致 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存活至关重要，并且 从线粒体到细胞质的细胞色素C释放诱导细胞死亡，我们假设这些 新发现的携带血红素蛋白与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期间宿主细胞反应的成分 IPD的病理生理学。分子健康中心杠杆的分子生理方法 下面提出了下面提出的疾病（MCHD）来评估这种创新的假设。在AIM 1中，我们将描述 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（S） 携带蛋白质。为了评估这一点，我们将使用可用的 蛋白质和线粒体细胞色素从人类永生和人类原发性分化中纯化 细胞。 SPN产生的H2O2引起的特定宿主细胞反应将通过靶向蛋白质组学研究， Western印迹，Elisa和Facs。整个转录组将进一步支持这些研究的证据 AIM 2将重点放在过氧化氢诱导的血红素携带的氧化的后果上 蛋白质。我们将使用与靶向蛋白质组学耦合的肺炎球菌肺炎的鼠标模型 RNA-seq研究以及组织学评估和超微结构显微镜研究，以研究 这种氧化反应对健康运输和肺部疾病的后果。鼠标模型 肺炎球菌疾病将用于评估一系列H2O2的清除剂的效率以降低 H2O2相关的肺炎球菌和/或侵入性疾病。这个高度创新的提议与 通过整合和利用Core B和Core C Technologies and Fardibilities和Core Core的目标， MCHD提供的资术和资源。