Regulation of the (poly) sialic virulence factor

（多）唾液酸毒力因子的调节

• 批准号: 6702853
• 负责人: Eric Ross Vimr
• 金额: $ 19.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702853
• 关键词: Escherichia coli infections; X ray crystallography; bacterial capsules; bacterial cytopathogenic effect; bioinformatics; disease /disorder etiology; enzyme activity; enzyme biosynthesis; enzyme structure; functional /structural genomics; gene expression; genetic regulation; host organism interaction; laboratory rat; mass spectrometry; matrix assisted laser desorption ionization; meningitis; nuclear magnetic resonance spectroscopy; paper chromatography; polymerase chain reaction; scintillation spectrometry; sialate; sialyltransferases; site directed mutagenesis; virulence

DESCRIPTION (provided by applicant): Bacterial sepsis and meningitis are the leading causes of mortality and hospitalization by infectious agents in the United States. Sepsis alone accounts for >700,000 life-threatening infections each year and over 200,000 deaths, with economic losses greatly exceeding one billion annually. Extraintestinal pathogenic Escherichia coli (ExPEC) are the predominant agents of these infections, with one serotype, E. coli O18:K1:H7, emerging as the leading cause of gram-negative neonatal meningitis and the most prevalent isolate from uncomplicated cystitis in women, thus making it a useful model of ExPEC infections in general. How ExPEC breach the host's mucosa and reach the systemic concentration (bacteremia) necessary for triggering sepsis and meningitis is not understood, although cell-surface modification with sialic acid is an important determinant. In this continuation application, we propose to complete our studies to determine how the sialic and polysialic acid virulence factors are synthesized, and then to identify other gene products required for systemic disease using a functional genomic approach. Specific Aim 1 will complete the analysis of sialic acid biosynthesis and the regulation of sialate metabolism. Preliminary data indicate that the synthesis of N-acetylmannosamine (ManNAc), the first committed step in sialate biosynthesis, does not involve ManNAc 6-phosphate. X-ray crystallography and NMR spectroscopy will complement the objectives of this aim. In Specific Aim 2, the structural basis for PSA biosynthesis will be determined by analyzing chimeric polysialyltransferases (polySTs) and using site-directed mutagenesis to understand catalytic mechanism. Our preliminary data indicate that these important enzymes are structurally and functionally distinct from mammalian polySTs, suggesting wide therapeutic potential for specific polyST inhibitors. Specific Aim 3 will apply signature-tagged mutagenesis to identify disease traits that are either distinct from PSA or function in concert with this capsule to define ExPEC virulence. Our objective in this aim is to establish the minimal systemic ExPEC "pathotype". The proposed studies have direct application to the development of new therapeutic approaches by identifying novel targets for non-antibiotic drug or vaccine design. New approaches are urgently needed to prevent or treat ExPEC infections in the rapidly aging US population, the very young and the increasingly large number of immunocompromised patients.

描述（由申请人提供）：细菌性败血症和脑膜炎是美国感染性病原体导致死亡和住院的主要原因。仅脓毒症每年就造成> 700，000例危及生命的感染和超过200，000例死亡，每年的经济损失大大超过10亿美元。肠外致病性大肠杆菌（ExPEC）是引起这些感染的主要病原体，其中有一种血清型，即E.大肠杆菌O 18：K1：H7，是革兰氏阴性新生儿脑膜炎的主要原因，也是女性单纯性膀胱炎中最常见的分离株，因此使其成为ExPEC感染的有用模型。ExPEC如何破坏宿主粘膜并达到引发败血症和脑膜炎所需的全身浓度（菌血症）尚不清楚，尽管唾液酸对细胞表面的修饰是一个重要的决定因素。在本延续申请中，我们建议完成我们的研究，以确定唾液酸和聚唾液酸毒力因子是如何合成的，然后使用功能基因组方法鉴定系统性疾病所需的其他基因产物。具体目标1将完成唾液酸生物合成的分析和唾液酸代谢的调节。初步数据表明，N-乙酰甘露糖胺（ManNAc）的合成，唾液酸生物合成的第一个关键步骤，不涉及ManNAc 6-磷酸。X射线晶体学和NMR光谱学将补充这一目标的目标。在具体目标2中，PSA生物合成的结构基础将通过分析嵌合聚唾液酸转移酶（polyST）和使用定点突变来理解催化机制来确定。我们的初步数据表明，这些重要的酶在结构和功能上不同于哺乳动物polyST，这表明特异性polyST抑制剂具有广泛的治疗潜力。具体目标3将应用签名标记的诱变来鉴定与PSA不同或与该胶囊协同作用的疾病性状，以定义ExPEC毒力。我们的目标是建立最小的系统性ExPEC“致病型”。拟议的研究通过确定非抗生素药物或疫苗设计的新靶点，直接应用于新治疗方法的开发。迫切需要新的方法来预防或治疗快速老龄化的美国人口、非常年轻和越来越多的免疫功能低下患者中的ExPEC感染。