Molecular basis of heme scavenging by Gram-positive bacteria

项目摘要 几乎所有种类的细菌都需要铁来生长，因为它是一种必需的金属辅因子， 微生物酶介导细胞代谢。在感染过程中，细菌病原体从 人血红蛋白（Hb），从红细胞中释放出来，含有人体血红蛋白的75-80%。 血红素形式的总铁含量（铁原卟啉IX）。本提案中概述的基础科学研究 将确定人类病原体白喉棒状杆菌如何从血红蛋白中获得铁。这项工作将有 广泛的影响，如C.白喉是放线菌门内的模式生物，其含有几种 作为人类病原体的细菌种类，以及作为人类病原体的主要组成部分的微生物。 胃肠道微生物组。研究将由一个已建立的研究者团队进行， 在微生物学、蛋白质组学、生物化学和结构生物学方面的互补专长。我们将确定 微生物受体如何捕获Hb并去除其血红素，以及细胞壁嵌入的蛋白质如何释放 血红素进入细胞。在目标#1中，我们将确定C。白喉使用HbpA受体捕获血红蛋白， 细胞表面和测试的假设，该受体的作品与表面相关血红素受体 扭曲血红蛋白并引发血红素释放在目标#2中，我们将确定血红素的分子基础， 通过确定保守区（CR）结构域在细胞壁中的分布范围， 放线菌通过形成低亲和力的转移复合物直接交换血红素。在目标#3中，我们将获得 应用质谱蛋白质组学方法对血红素摄取过程的系统水平理解 以确定每种成分的丰度和位置，并通过开发和应用一种新的荧光 Hb-报告子追踪细胞培养物中血红素从Hb的去除。这些研究将使我们能够定量评估 每个系统组件在血红素摄取的重要性，并测试假设，他们形成一个分子 血红素通过它流到细胞膜上。这些研究的结果将提供基本的见解 如何C。白喉杆菌和其他放线菌获得血红素铁，并开发出可推广的工具来研究 这个过程在活细菌中进行。结合起来，这项研究的结果可能会导致新的疗法来治疗 由抗生素耐药细菌引起的感染，通过破坏血红素输入起作用。

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.