Iron Acquisition From Host Proteins in S. pyogenes

描述（由申请人提供）：该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。本研究特别关注化脓性链球菌在感染过程中获取铁的分子机制。大多数细菌都需要铁，铁是电子转移的催化剂，也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，导致一个基本上没有未结合铁的环境，这反过来又给需要铁来生长的入侵细菌带来了挑战。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，化脓链球菌和相关病原体捕获和运输血红素或铁的机制尚未得到很好的表征。在这个实验室进行的遗传研究发现了两个铁调控的操纵子，分别是sia（链球菌铁获取）和sit（链球菌铁运输），它们参与血红蛋白的利用。第一个具体目的是剖析sia和sit操纵子的功能及其在化脓性葡萄球菌生理中的作用。遗传和生化分析，包括突变特征和体内结合和运输分析将被使用。第二个具体目的是分析操纵子在毒力中的作用；研究将在斑马鱼和小鼠感染模型中进行。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捕获将通过体外结合试验进行研究；定点诱变和任意PCR诱变将用于鉴定功能性受体结构域。不同受体组分之间的相互作用将通过固相结合试验、交联和免疫沉淀来研究。从宿主血红蛋白中获取铁可能对化脓性链球菌（一种专性人类寄生虫）的生理和毒力有重要影响。提出的化脓性葡萄球菌铁获取特性将促进我们对这一重要病原体的理解，并将增加对其他革兰氏阳性细菌铁摄取过程的理解，其中许多革兰氏阳性细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.