Molecular basis of heme scavenging by Gram-positive bacteria

革兰氏阳性菌清除血红素的分子基础

• 批准号: 10330038
• 负责人: Robert Thompson Clubb
• 金额: $ 60.69万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-19 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330038
• 关键词: Actinobacteria class; Affinity; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Basic Science; Bifidobacterium; Binding; Biochemical; Biochemistry; Biology; Blood; Cell Culture Techniques; Cell Wall; Cell surface; Cells; Cellular Membrane; Chemicals; Complement 3d Receptors; Complex; Corynebacterium diphtheriae; Cytoplasm; Development; Diphtheria; Enzymes; Erythrocytes; Etiology; Excision; Goals; Gram-Positive Bacteria; Harvest; Heme; Heme Iron; Hemoglobin; Human; Human body; Infection; Infectious Skin Diseases; Iron; Learning; Location; Mass Spectrum Analysis; Mediating; Membrane; Metabolism; Metals; Methods; Microbe; Microbiology; Molecular; Mycobacterium tuberculosis; NMR Spectroscopy; Nutritional Immunity; Pathogenicity; Process; Proteins; Proteomics; Reporter; Research; Research Personnel; Research Project Grants; Structural Biochemistry; Surface; System; Testing; Upper Respiratory Infections; Work; X-Ray Crystallography; cofactor; experimental study; extracellular; gut microbiome; gut microbiota; haptoglobin-hemoglobin complex; heme receptor; human pathogen; insight; iron protoporphyrin IX; member; microbial; model organism; novel; novel therapeutics; pathogen; pathogenic bacteria; protein function; receptor; sensor; structural biology; tool; uptake

Project Summary Nearly all species of bacteria require iron to grow because it is an essential metal cofactor that is used by microbial enzymes to mediate cellular metabolism. During infections, bacterial pathogens forage iron from human hemoglobin (Hb) that is released from red blood cells, which contains ∼75–80% of the human body's total iron content in the form of heme (iron protoporphyrin IX). The basic science studies outlined in this proposal will determine how the human pathogen Corynebacterium diphtheriae acquires iron from Hb. This work will have a broad impact, as C. diphtheriae is a model organism within the Actinobacteria phylum, which contains several species of bacteria that are human pathogens, as well as microbes that are major components of the human gastrointestinal microbiome. Research will be performed by an established team of investigators that have complementary expertise in microbiology, proteomics, biochemistry and structural biology. We will determine how microbial receptors capture Hb and remove its heme, and how cell wall embedded proteins ferry released heme into the cell. In aim #1, we will determine how C. diphtheriae uses the HbpA receptor to capture Hb on the cell surface and test the hypothesis that the receptor works in concert with surface associated heme-receptors to distort Hb and trigger heme release. In aim #2, we will determine the molecular basis through which heme is passed across the cell wall by determining how widely distributed Conserved Region (CR) domains in Actinobacteria directly exchange heme by forming low-affinity transfer complexes. In aim #3, we will obtain a systems-level understanding of the heme uptake process by applying mass spectrometry proteomics methods to determine each component’s abundance and location, and by developing and applying a novel fluorogenic Hb-reporter to track heme removal from Hb in cell culture. These studies will enable us to quantitatively assess the importance of each system component in heme uptake, and to test the hypothesis that they form a molecular wire through which heme flows to the membrane. The results of these studies will provide fundamental insight into how C. diphtheriae and other Actinobacteria acquire heme-iron, and develop generalizable tools to study this process in live bacteria. Combined, the results of this research could lead to new therapeutics to treat infections caused by antibiotic resistant bacteria that work by disrupting heme import.

项目摘要 几乎所有种类的细菌都需要铁生长，因为它是一种必需的金属辅助因子，由 微生物酶介导细胞代谢。在感染期间，细菌病原体觅食铁 从红细胞释放的人类血红蛋白（HB），其中约有75-80％的人体 血红素（铁原磷脂IX）形式的总铁含量。该提案中概述的基础科学研究 将确定人类病原体甲状腺杆菌如何从HB中获取铁。这项工作将有 巨大的影响，因为白喉梭菌是静脉细菌中的模型生物，其中包含几个 是人类病原体的细菌，以及人类主要成分的微生物 胃肠道微生物组。研究将由一个成熟的调查员团队进行 微生物学，蛋白质组学，生物化学和结构生物学方面的补充专业知识。我们将确定 微生物接收器如何捕获HB并去除血红素，以及细胞壁嵌入蛋白渡轮如何释放 血红素进入细胞。在AIM＃1中，我们将确定C. diphtheriae如何使用HBPA受体捕获HB 细胞表面并检验该受体与表面相关的血红素受体一起工作的假设 扭曲HB并触发血红素释放。在AIM＃2中，我们将确定血红素是的分子基础 通过确定分布广泛的保守区域（Cr）域的跨越细胞壁的传递 静脉细菌通过形成低亲和力转移复合物直接交换血红素。在AIM＃3中，我们将获得一个 通过应用质谱蛋白质组学方法，系统级别对血红素摄取过程的了解 确定每个组件的抽象和位置，并通过开发和应用新颖的氟化 HB重复蛋白跟踪细胞培养中HB的血红素去除。这些研究将使我们能够定量评估 每个系统成分在血红素摄取中的重要性，并测试它们形成分子的假设 线血红素流到膜的电线。这些研究的结果将提供基本的见解 在白喉梭菌和其他肌动杆菌如何获得血红素铁和开发可概括的工具来研究 活细菌中的这个过程。合并，这项研究的结果可能导致新的治疗疗法 由抗生素耐药细菌引起的感染，通过破坏血红素进口而起作用。