Iron Acquisition From Host Proteins in S. pyogenes

描述(由申请人提供)：这个项目的长期目标是增加对人类病原体化脓性链球菌的生理学和毒力的了解。这项研究特别涉及化脓性链球菌在感染过程中获取铁的分子机制。大多数细菌需要铁，铁是电子转移的催化剂，是许多重要酶的基本成分。哺乳动物体内的绝大多数铁被高亲和力蛋白质隔离，导致环境基本上没有游离铁，这反过来又对入侵的细菌构成了挑战，这些细菌需要铁来生长。血红素和血红素化合物是溶血性链球菌铁的重要来源。然而，化脓性链球菌和相关病原体用来捕获和运输血红素或铁的机制还没有得到很好的描述。该实验室进行的遗传学研究确定了两个铁调节操纵子，名为SIA(用于链球菌铁获取)和SIT(用于链球菌铁运输)，它们参与了对血红蛋白的利用。第一个特定目的是剖析SIA和SIT操纵子的功能及其在化脓性链球菌生理学中的作用。将使用包括突变体特征以及采用体内结合和转运分析的遗传和生化分析。第二个具体目的是分析操纵子在毒力中的作用；将在斑马鱼和小鼠感染模型中进行研究。第三个具体目的是分析参与血红蛋白利用的表面受体的结构和功能。底物识别和血红素或铁的捕获将通过体外结合分析进行研究；定点突变和随机聚合酶链式反应突变将用于识别功能受体结构域。不同受体组分之间的相互作用将通过固相结合分析、交联法和免疫沉淀进行研究。从宿主血红素蛋白中获取铁可能对化脓性链球菌的生理和毒力有重要影响，化脓性链球菌是人类专性寄生虫。化脓性链球菌铁摄取特性的提出将促进我们对这一重要病原体的了解，并将有助于理解其他革兰氏阳性菌摄铁的过程，其中许多革兰氏阳性菌是重要的人类病原体。

DESCRIPTION (provided by applicant): The long-term goal of this project is to increase the understanding of the physiology and virulence of the human pathogen Streptococcus pyogenes. This investigation is specifically concerned with the molecular mechanisms used by S. pyogenes to obtain iron during infection. Most bacteria require iron, which serves as a catalyst for electron transfer and is an essential component of many important enzymes. The vast majority of iron in the mammalian body is sequestered by high affinity proteins, resulting in an environment that is essentially free of unbound iron, which in turn presents a challenge to invading bacteria that need iron for growth. Heme and heme-compounds are valuable sources of iron for the hemolytic Streptococcus. Yet, the mechanisms used by S. pyogenes and related pathogens to capture and transport heme or iron are not well characterized. Genetic studies done in this laboratory identified two iron-regulated operons named sia (for Streptococcal Iron Acquisition) and sit (for Streptococcal Iron Transport) that are involved in the utilization of hemoglobin. The first specific aim dissects the function of the sia and the sit operons and their role in S. pyogenes physiology. Genetic and biochemical analysis that includes mutant characterization and employs in vivo binding and transport assays will be used. The second specific aim analyzes the operon role in virulence; studies will be conducted in zebrafish and mice infection models. The third specific aim analyzes the structure and function of surface receptors involved in hemoprotein utilization. Substrate recognition and heme or iron capture will be investigated by in vitro binding assays; site-directed mutagenesis and arbitrary PCR mutagenesis will be used to identify functional receptor domains. The interactions between different receptor components will be studied with solid phase binding assays, cross-linking, and immunoprecipitation. Iron acquisition from host hemoproteins is likely to have important implications on the physiology and virulence of S. pyogenes, an obligate human parasite. The proposed characterization of iron acquisition in S. pyogenes will advance our understanding of this important pathogen, and will add to the understanding of the process of iron uptake in other Gram-positive bacteria, many of which are important human pathogens.