STREPTOCOCCAL HYALURONATE CAPSULE

链球菌透明质酸胶囊

基本信息

批准号：
2886979
负责人：
IVO J VAN DE RIJN
金额：
$ 17.79万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2001-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2886979
关键词：
DNA footprinting Escherichia coli RNase protection assay Streptococcus pyogenes bacterial capsules bacterial genetics carbohydrate biosynthesis gel mobility shift assay gene expression genetic regulatory element hyaluronate ligase molecular cloning northern blottings nucleic acid sequence operon transcription factor western blottings

项目摘要

DESCRIPTION (Adapted from the Applicant's Abstract): Lancefield group A streptococci (Streptococcus pyogenes) are one of the most prevalent human pathogens (25 million infection/year) capable of causing a variety of severe suppurative infections, as well as non-suppurative sequelae such as post-streptococcal glomerulonephritis and rheumatic fever. In addition, group A streptococci were shown to induce a lethal disease that resembles toxic shock syndrome. Within the last decade, an increase in the number of invasive group A streptococcal infections associated with bacteremia deep soft-tissue infection, shock, and multi- organ failure were observed in patients with no prior underlying disease. Group A and C streptococci have the ability to surround themselves with a capsule formed by hyaluronic acid which serves as a virulence factor. The structure of streptococcal hyaluronic acid is identical to mammalian hyaluronic acid where it is an ubiquitous component of connective tissue. The overall objective of this research program is to continue a systematic study of the Streptococcus pyogenes hyaluronic acid synthase complex in order to better understand the mechanism of expression of this capsulae in the human host. Towards this objective they plan to : 1) Isolate and sequence the hyaluronate synthase (has) capsule operon. Determine which genes of the operon are required for production of capsule in group A streptococci and analyze group A and non-group A streptococci for these genes; 2) determine the function of the genes required for hyaluronic acid capsule biosynthesis. Overexpress and purify the has A product to determine whether it is the intact synthase or a component thereof; 3) determine whether the has operon transcript is regulated via transcriptional or post- transcriptional mechanisms. Dot-blot analyses, Northern blots, and RNAase protection assays will be used to resolve the amount and size of has operon mRNA present in strains of group A streptococci during various stages of growth; and, 4) analyze the cis and trans elements which regulate the has operon. Promoter sequence(s) and transcription accessory factors will be determined by sequencing of the 1 kb region upstream of the has A promoter, and DNAase 1 footprinting of the promoter region. If post-transcriptional mechanisms regulate the expression of the hyaluronic capsule, factors such as mRNA stability, the presences of inhibitory enzymes, or mutations within the operon will be investigated. The completion of this aims will also provide investigators with a biochemical analysis of the synthase complex and a better understanding of the expression of this important bacterial and mammalian mucopolysaccharide.

描述（改编自申请人的摘要）：Lancefield Group 链球菌（链球菌为链球菌）是最普遍的人类病原体（2500万感染/年）能够引起多种严重的化脓感染以及非支持后遗症例如链球菌后肾小球肾炎和风湿热。在此外，A组链球菌被证明会诱导致命疾病这类似于有毒休克综合征。在过去的十年中，侵入性A链球菌感染的数量增加与细菌血症深软组织感染，休克和多种多样有关在没有先验基础的患者中观察到器官衰竭疾病。 A组和C链球菌具有包围的能力自己用透明质酸形成的胶囊，用作毒力因子。链球菌透明质酸的结构是与哺乳动物透明质酸相同，它无处不在结缔组织的成分。这项研究的总体目标程序将继续对化脓性链球菌进行系统研究透明质酸合酶复合物，以便更好地了解人类宿主中这种囊肿表达的机理。向他们计划的这个目标：1）隔离并对透明质酸合酶（HAS）胶囊操纵子。确定操纵子的哪个基因在A组链球菌中生产胶囊的必需品并分析这些基因的A组和非组A链球菌； 2）确定透明质酸胶囊生物合成所需的基因的功能。过表达并净化该产品以确定是否是完整的合酶或其组件； 3）确定是否有操纵子的转录本通过转录或后的转录进行调节转录机制。点印刷分析，北印迹和 RNAase保护分析将用于解决在A组链球菌中存在操纵子mRNA 各个阶段的增长阶段； 4）分析顺式和反式元素调节有操纵子。启动子序列和转录辅助因子将通过测序1 kb区域确定具有启动子的上游和启动子的DNAase 1足迹地区。如果转录后机制调节了透明质胶囊，诸如mRNA稳定性之类的因素，这些因素抑制酶或操纵子内的突变将是调查。此目标的完成也将提供对合酶复合物和A的生化分析的研究者更好地理解这种重要细菌和哺乳动物粘多糖。