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Regulation of the Poly Sialic Virulence Factor

多唾液酸毒力因子的调节

基本信息

批准号：
8063545
负责人：
Eric Ross Vimr
金额：
$ 23.54万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8063545
关键词：
Acetylation Acetylesterase Acetyltransferase Acids Acylneuraminate Cytidylyltransferase Anabolism Animal Model Antibiotic Resistance Antigens Bacteria Bacterial Antigens Bacterial Meningitis Bacteriophages Binding Biochemical Biochemical Genetics Campylobacter Carbohydrates Catabolism Cell surface Cessation of life Chemical Structure Communicable Diseases Complex Coupled Coupling Defense Mechanisms Detection Disease Effectiveness Enzymes Escherichia coli Esters Event Genes Genetic Goals Health Care Costs Helicobacter Hemophilus Human Hybrids Immune Immunocompromised Host Incidence Infection Investigation Link Membrane Meningitis Meningococcal vaccine Metabolism Methodology Methods Microbe Microbial Genetics Microscopic Modification Molecular Mutagenesis N-Acetylneuraminic Acid Natural Immunity Neisseria meningitidis Neonatal Pathogenesis Pathway interactions Pharmacotherapy Phase Polymerase Polysaccharides Polysialic Acid Population Prevention strategy Preventive Proteins Publishing Quantum Dots Regulation Relative (related person)Research Support Rest Role Screening procedure Sepsis Sialic Acids Site Streptococcus Group B Structure Surface Systemic disease Techniques Technology Testing Therapeutic United States Vaccines Variant Virulence Virulence Factors Virulent aged base capsule design esterase fungus human disease in vivo innovation mutant novel novel strategies novel therapeutics pathogen pathogenic Escherichia coli periplasm polymerization polysialic acid O-acetyltransferase public health relevance therapeutic development treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Extraintestinal pathogenic Escherichia coli (ExPEC) are major pathogens in mostly the very young, aged or immunocompromised human population resulting in tens of thousands of deaths each year and billions of dollars in healthcare costs. For example, the predominant ExPEC subtype, E. coli O18:K1, expresses O18 somatic and K1 polysialic acid capsule antigens as two major virulence factors responsible for making these strains the leading causes of neonatal bacterial meningitis. Using microbial genetics and innovative methods of carbohydrate analysis, we defined the complete pathways for sialic acid transport, synthesis, polymerization, catabolism and modification of both monomeric and polysialic acids. The modification mechanism was linked to a novel bacterial virus (CUS-3) carrying the phase-variable acetylase gene catalyzing stochastic O-acetylation of the sialic acid exocyclic chain. We hypothesize that understanding the in vivo functions of these bacterial antigens will facilitate approaches targeting them for new therapeutic development. We have designed three specific aims to test this hypothesis: 1) Determine the contribution of monomeric sialic acid O- acetylation to overall polysialic acid modification by genetically altering the acetyltransferase, NeuD, and acetylesterase, NeuA* (NeuA-star). This aim focuses on the enzymes involved in the reciprocal addition (NeuD) or subtraction (NeuA*) of O- acetyl esters to or from monomeric sialic acids prior to their incorporation into polysialic acid. 2) Determine the dynamics of capsular polysialic acid modification in vivo using an innovative flow cytometric technique and microscopic methods to distinguish between acetylated and unacetylated phases in different host compartments. 3) Determine the molecular basis for coupling polysaccharide synthesis to export by (i) constructing in frame deletions of all region 1 export genes, (ii) determining whether 3-deoxy-D-manno- octulosonate is required for biosynthesis, (iii) identifying the polymerase domain(s) interacting with the accessory protein, KpsC, (iv) using Quantum-dot technology and K1- specific phage to determine the site of capsule export, and (v) determining the chemical structure of the initiation complex. PUBLIC HEALTH RELEVANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) causes tens of thousands of deaths in the United States each year, and is associated with billions of dollars in healthcare costs. The increasing incidence of antibiotic resistance coupled with the lack of safe and effective vaccines or other drug therapies supports research effort directed toward identification of bacterial targets for new therapeutic development. Our renewal application is focused on the acetylated form of the E. coli K1 polysialic acid capsular polysaccharide, present in all of the most virulent K1 strains, and the fundamental machinery for capsular polysaccharide biosynthesis producing the major virulence factor in the majority of ExPEC strains.

描述（由申请人提供）：肠外致病性大肠杆菌 (ExPEC) 是大多数幼儿、老年人或免疫功能低下人群的主要病原体，每年导致数万人死亡和数十亿美元的医疗费用。例如，主要的 ExPEC 亚型大肠杆菌 O18:K1 表达 O18 体细胞抗原和 K1 聚唾液酸荚膜抗原，作为两种主要毒力因子，使这些菌株成为新生儿细菌性脑膜炎的主要原因。利用微生物遗传学和碳水化合物分析的创新方法，我们定义了单体和聚唾液酸的唾液酸运输、合成、聚合、分解代谢和修饰的完整途径。该修饰机制与一种携带相变乙酰化酶基因的新型细菌病毒（CUS-3）有关，该基因可催化唾液酸环外链的随机 O-乙酰化。我们假设了解这些细菌抗原的体内功能将有助于针对它们的新治疗方法的开发。我们设计了三个具体目标来检验这一假设：1) 通过基因改变乙酰转移酶 NeuD 和乙酰酯酶 NeuA* (NeuA-star)，确定单体唾液酸 O-乙酰化对整体聚唾液酸修饰的贡献。该目标集中于在将单体唾液酸掺入聚唾液酸之前参与 O-乙酰酯与单体唾液酸的相互加成 (NeuD) 或减去 (NeuA*) 的酶。 2) 使用创新的流式细胞术技术和显微方法确定体内荚膜聚唾液酸修饰的动力学，以区分不同宿主区室中的乙酰化和非乙酰化相。 3) 通过以下方式确定耦合多糖合成与输出的分子基础：(i) 构建所有区域 1 输出基因的框内删除，(ii) 确定生物合成是否需要 3-脱氧-D-甘露糖辛酸，(iii) 识别与辅助蛋白 KpsC 相互作用的聚合酶结构域，(iv) 使用量子点技术和 K1-特异性噬菌体确定胶囊出口的位点，以及(v)确定起始复合物的化学结构。公共卫生相关性：肠外致病性大肠杆菌 (ExPEC) 每年在美国导致数万人死亡，并造成数十亿美元的医疗费用。抗生素耐药性的发生率不断增加，加上缺乏安全有效的疫苗或其他药物疗法，支持了旨在识别细菌靶点以开发新疗法的研究工作。我们的更新应用集中于大肠杆菌 K1 聚唾液酸荚膜多糖的乙酰化形式，存在于所有毒性最强的 K1 菌株中，以及荚膜多糖生物合成的基本机制，在大多数 ExPEC 菌株中产生主要毒力因子。