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

描述（由申请人提供）：拟议的项目是一个正在进行的努力，以了解细菌病原体的血红素吸收和运输所发挥的作用的一部分。这项工作的长期目标是阐明的机制参数，支配血红素之间的吸收和转运蛋白转移的特异性和有效性。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。血红素摄取和/或转运的抑制可以为耐药性感染提供替代治疗。 该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。这将通过停流分光光度法和冷冻淬灭共振拉曼（Raman）光谱法结合定点诱变来实现。这种关注的原因是这些蛋白质进行血红素内化的第一步。因此，如果铁同化的血红素依赖性机制通过临床干预被抑制，这些受体似乎是最好的第一个目标，因为它们位于细胞表面。在我们的机制研究中靶向的受体是分别来自志贺氏菌和粘质沙雷氏菌的TonB依赖性ShuA和SmHasR受体。拟议项目的具体目标是：1。阐明血红素摄取到ShuA的细胞外结合口袋中的机制细节。 ShuA已经被证明可以从血红蛋白中摄取血红素，我们最近也证明了它可以结合游离血红素。血红素摄取率和机制ShuA（WT）和它的保守的His突变体从这两个来源将进行比较和对比，努力建立轴向配体交换步骤的顺序和量化的机制的重要性，蛋白质-蛋白质复合的效率加载的细胞外结合口袋。2.阐明血红素摄取到HasR的细胞外结合口袋中的机制细节。 ITC已经证明Has R从其同源分泌的血红素HasA中摄取血红素，并结合游离血红素。以类似于ShuA所述的方式，将比较和对比这些反应的动力学，以阐明HasA-HasR缔合对血红素同化的第一步效率的重要性。 公共卫生相关性：人类和动物宿主的细菌感染继续直接或通过其对食品供应的有害影响破坏全世界的公共卫生。在宿主中建立感染的决定性因素之一是铁的可用性。许多细菌病原体产生的蛋白质可以利用宿主血液中大量的血红素（含铁色素）来获得铁。这一优势是通过从宿主蛋白质中切除血红素、内化血红素并切除铁来实现的。重要的是，如果血红素同化系统受损，这些生物体可能会因铁而挨饿。由于铁饥饿破坏了它们定殖宿主的能力，因此对它们的获取和血红素的细胞内转运至关重要的蛋白质是针对细菌感染的预防性和/或治疗性治疗的潜在靶标。这项研究旨在通过详细了解这些蛋白质的功能，为靶向这些蛋白质奠定基础。

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.