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，因为它是一种必不可少的金属辅因子，被 调节细胞代谢的微生物酶。在感染期间，细菌病原体从 人的血红蛋白(Hb)是从红细胞中释放出来的，它含有人体∼的75%-80% 以血红素形式表示的总铁含量(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Hb获得铁。这项工作将会有 广泛的影响，因为白喉杆菌是放线菌门中的模式生物，它包含几个 作为人类病原体的细菌种类，以及作为人类主要组成部分的微生物 胃肠道微生物组。研究将由一个成熟的调查团队进行，该团队拥有 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业知识。我们将决定 微生物受体如何捕获Hb并清除其血红素，以及细胞壁嵌入的蛋白质如何释放 亚铁血红素进入细胞。在目标1中，我们将确定白喉隐翅虫是如何利用HBPA受体捕获Hb的。 并测试受体与表面相关的血红素受体协同工作的假设 扭曲Hb并引发血红素释放。在目标2中，我们将确定血红素通过的分子基础 通过确定保守区域(CR)结构域在细胞壁上的分布 放线菌通过形成低亲和力的转移复合体直接交换血红素。在目标3中，我们将获得一个 应用质谱仪蛋白质组学方法从系统水平理解血红素摄取过程 确定每个组分的丰度和位置，并通过开发和应用一种新的荧光素 HB-记者追踪细胞培养中Hb中的血红素去除。这些研究将使我们能够定量评估 每个系统成分在血红素吸收中的重要性，并检验它们形成分子的假设 金属丝，通过它，血红素流到膜上。这些研究的结果将提供基本的见解 了解白喉杆菌和其他放线菌是如何获得血红素铁的，并开发出可推广的工具来研究 这一过程在活细菌中进行。总而言之，这项研究的结果可能会导致治疗的新疗法 由抗药性细菌引起的感染，这些细菌通过扰乱血红素进口而发挥作用。

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.