Elucidating the Mechanism of Heme Transport across the Outer Membrane of Gram-neg

描述（由申请人提供）：拟议的项目是正在进行的努力的一部分，旨在了解细菌病原体对血红素的摄取和运输所起的作用。这项工作的长期目标是阐明控制血红素在摄取和运输蛋白之间转移的特异性和有效性的机制参数。这项工作与人类健康的相关性在于它有可能产生治疗细菌感染的新策略。抑制血红素摄取和/或运输可能为抗生素耐药感染提供替代治疗。本项目的下一阶段将集中于阐明两类外膜血红素受体的机制细节。这将通过停止流动分光光度法和冷冻猝灭共振拉曼（FQrR）光谱结合定点诱变来实现。之所以如此关注，是因为这些蛋白质是血红素内化的第一步。因此，如果通过临床干预抑制血红素依赖的铁同化机制，这些受体似乎是最佳的首选靶点，因为它们位于细胞表面。在我们的机制研究中，靶向受体分别是来自痢疾志贺氏菌和粘质沙雷氏菌的TonB依赖性的shu和SmHasR受体。拟建项目的具体目标是：1。阐明血红素摄取到细胞外结合袋的机制细节。研究表明，舒亚酶可以从血红蛋白中提取血红素，我们最近也发现，舒亚酶可以结合游离血红素。将比较和对比这两种来源的joshua （WT）及其保守突变体对血红素摄取的速率和机制，以建立轴向配体交换步骤的顺序，并量化蛋白质-蛋白质络合在细胞外结合袋加载效率中的机制重要性。2. 阐明血红素摄取到HasR细胞外结合袋的机制细节。ITC显示R可以从其同源的分泌血红素ha中提取血红素，并结合游离血红素。以一种类似于描述shu的方式，这些反应的动力学将被比较和对比，以阐明HasA-HasR结合对血红素同化第一步效率的重要性。

DESCRIPTION (provided by applicant): The proposed project is part of an ongoing effort to understand the roles played by heme uptake by and transport within bacterial pathogens. The long-term goal of this work is to elucidate the mechanistic parameters that govern the specificity and efficacy of heme transfer among the uptake and transport proteins. The relevance of this work to human health lies in its potential to spawn new strategies for treatment of bacterial infections. Inhibition of heme uptake and/or transport could provide alternative treatments for antibiotic-resistant infections. The next phase of this project will focus on elucidating the mechanistic details of two classes of outer membrane heme receptors. This will be accomplished through a combination of stopped flow spectrophotometry and freeze-quench resonance Raman (FQrR) spectroscopy in conjunction with site directed mutagenesis. The reason for this focus is that these proteins carry out the first step toward internalization of heme. Thus, if heme-dependent mechanisms for iron assimilation are to be inhibited via clinical intervention, these receptors would appear to be the best first targets as they reside at the cell surface. The receptors to be targeted in our mechanistic studies are the TonB- dependent ShuA and SmHasR receptors from Shigella dysenteriae and Serratia marcescense, respectively. The specific aims of the proposed project are: 1. to elucidate the mechanistic details of heme uptake into the extracellular binding pocket of ShuA. ShuA has been shown to take heme from hemoglobin and we have recently shown that it binds free heme. Rates and mechanisms of heme uptake by ShuA(WT) and its conserved His mutants from these two sources will be compared and contrasted in an effort to establish the order of axial ligand exchange steps and quantify the mechanistic importance of protein-protein complexation in the efficiency of loading the extracellular binding pocket. 2. to elucidate the mechanistic details of heme uptake into the extracellular binding pocket of HasR. Has R has been shown by ITC to take heme from its cognate secreted hemophore, HasA, and to bind free heme. In a fashion similar to that described for ShuA, the kinetics of these reactions will be compared and contrasted in order to shed light on the importance of the HasA-HasR association to the efficiency of this first step in heme assimilation. PUBLIC HEALTH RELEVANCE: Bacterial infections in human and animal hosts continue to undermine public health the world over, both directly and through their deleterious effects on the food supply. Among the deterministic factors for establishing infection in the host is the availability of iron. Many bacterial pathogens produce proteins that tap the huge pool of heme, the iron-containing pigment in the blood of the host, to acquire iron. This advantage is taken by excising heme from host proteins, internalizing it, and excising the iron. Importantly, these organisms can starve for iron if their heme assimilation system is compromised. As iron starvation undermines their ability to colonize the host, the proteins critical to their acquisition and intracellular transport of heme are potential targets for prophylactic and/or therapeutic treatments against bacterial infection. This study aims to lay a foundation for targeting these proteins by building a detailed understanding of how they function.