Heme Acquisition in Shigella Dysenteriae.

描述(由申请人提供)：当前提案的目标是 在功能和结构水平上鉴定和表征蛋白质 参与细菌病原体中的血红素运输。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 在一定程度上与它们的毒力有关。体内铁的主要来源是 络合在亚铁血红素和含亚铁血红素的蛋白质中。病原菌有 开发了复杂的机制，它们通过这种机制从宿主那里获取铁 血红素和血红素蛋白。目前发生的增加的 抗生素耐药菌株产生了开发替代品的需要 针对这类病原体的策略。结构和功能表征 参与血红素运输和储存的蛋白质应该使其有可能 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(HUTA)与周质血红素结合蛋白 (关闭)，将允许对血红素运输蛋白进行生化表征 在革兰氏阴性病原体中。这项研究将针对两个具体的 目的：第一，血红素与受体结合的性质和转运 第二，血红素吸收的机制。定点突变 与光学吸收、荧光、MCD等物理技术相结合 而共振拉曼光谱将提供关于 血红素结合和运输所需的结构特征。这个 外膜受体(HutA)和周质膜的底物特异性 结合蛋白(SHORT)将用取代的卟啉测定，用 前提是这类数据可能会为小型企业的开发提供模板 分子抑制剂。HutA在一株血红素缺陷型E. Coli允许我们直接监测血红素摄取作为细胞活力的函数 并提供了一种直接确定位点定向的血红素转运的方法。 诱变研究。此外，细菌血红素加氧酶的共表达 随着Huta受体的发展，将成为一种快速比色屏幕 未来的定点突变和抑制剂筛选。它的氧化还原性质 血红素的运输也将用氧化还原失活的非铁进行调查 金属卟啉，如钴和锌原卟啉IX。曾经的理解是 血红素在细菌病原体中的转运机制已被确立 将为未来设计选择性的血红素转运抑制剂奠定基础。

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.