Heme Acquisition in Shigella Dysenteriae.

描述（申请人提供）：本提案的目的是 在功能和结构水平上鉴定和表征蛋白质， 参与细菌病原体中血红素的运输。致病菌需要 铁对它们的生存和生长以及获得这种元素的能力是至关重要的。 部分与其毒性有关。体内铁的主要来源是 与血红素和含血红素的蛋白质复合。病原菌有 发展出复杂的机制， 血红素和血红素蛋白。目前， 抗药性菌株产生了开发替代药物的需求， 针对此类病原体的策略。结构和功能表征 蛋白质参与血红素的运输和储存， 开发特定的抑制剂。目的基因的表达和纯化 外膜血红素受体和周质血红素结合蛋白 （ShuT），将允许血红素转运蛋白的生化表征， 革兰氏阴性病原体研究将针对两个具体的 目的：一是血红素与受体结合和转运的性质 蛋白质，第二，血红素摄取的机制。定点诱变 与物理技术如光学吸收、荧光、MCD 和共振拉曼分光光度法将提供关于 血红素结合和运输所需的结构特征。的 外膜受体（HutA）和周质受体的底物特异性 结合蛋白（ShuT）将用取代的卟啉测定， 前提是这些数据可以为开发小型 分子抑制剂。HutA基因在血红素缺陷型大肠杆菌中的表达。 大肠杆菌使我们能够直接监测血红素吸收作为细胞活力的函数 并提供了一种直接的方法， 诱变研究。此外，细菌血红素加氧酶的共表达 与HutA受体将被开发为一个快速比色屏幕， 未来的定点诱变和抑制剂筛选。的氧化还原性质 还将使用氧化还原非活性非铁研究血红素运输 金属卟啉如Co和Zn-原卟啉IX。一旦了解了 细菌病原体中血红素转运的机制已经建立， 将为未来设计血红素转运选择性抑制剂奠定基础。

DESCRIPTION (Provided by Applicant): The objective of the current proposal is to identify and characterize at a functional and structural level the proteins involved in heme transport in bacterial pathogens. Pathogenic bacteria require iron for their survival and growth and the ability to acquire this element is linked in part to their virulence. The major source of iron within the body is complexed in heme and heme-containing proteins. Pathogenic bacteria have developed sophisticated mechanisms by which they acquire iron from the hosts heme and hemeproteins. The current increase in the occurrence of antibiotic-resistant strains has created a need to develop alternative strategies to target such pathogens. Structural and functional characterization of proteins involved in heme transport and storage should make it possible to develop specific inhibitors. The expression and purification of the outer-membrane heme receptor (HutA) and periplasmic heme-binding protein (ShuT), will allow biochemical characterization of the heme transport proteins in gram-negative pathogens. The study will be directed towards two specific goals: first, the nature of heme binding to the receptor and transport proteins, and second, the mechanism of heme uptake. Site-directed mutagenesis together with physical techniques such as optical absorption, fluorescence, MCD and resonance Raman spectrophotometry will provide information on the structural features required for heme-binding and transport. The substrate-specificity of the outer-membrane receptor (HutA) and the periplasmic binding protein (ShuT) will be determined with substituted porphyrins, with the premise that such data may provide templates for the development of small molecule inhibitors. The expression of HutA in a heme deficient strain of E. coli allows us to directly monitor heme uptake as a function of cell viability and provides a direct method of determining heme transport in site-directed mutagenesis studies. In addition the coexpression of a bacterial heme oxygenase with the HutA receptor will be developed as a rapid colorimetric screen for future site-directed mutagenesis and inhibitor screening. The redox nature of heme transport will also be investigated with redox inactive non-iron metalloporphyrins such as Co and Zn-protoporphyrin IX. Once an understanding of the mechanism of heme transport in bacterial pathogens has been established the stage will be set for future design of selective inhibitors of heme transport.