Regulation of the Poly Sialic Virulence Factor

多唾液酸毒力因子的调节

• 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
• 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

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(申请人提供)：肠外致病性大肠埃希氏菌(ExPEC)是主要病原体，主要发生在非常年轻、老年或免疫功能低下的人群中，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E.ColiO18：K1表达O18体细胞和K1聚唾液酸囊膜抗原，这是导致这些菌株成为新生儿细菌性脑膜炎的主要原因的两个主要毒力因子。利用微生物遗传学和碳水化合物分析的创新方法，我们定义了唾液酸运输、合成、聚合、分解代谢和修饰单体和多唾液酸的完整途径。其修饰机制与一种新型细菌病毒(CUS-3)相连，该病毒携带可变相变乙酰酶基因，催化唾液酸外环链的随机O-乙酰化。我们推测，了解这些细菌抗原的体内功能将有助于针对它们进行新的治疗开发。我们设计了三个特定的目标来验证这一假设：1)通过遗传改变乙酰基转移酶NeUD和乙酰酯酶NeuA*(NeuA-STAR)，确定单体唾液酸O-乙酰化对整个聚唾液酸修饰的贡献。这一目标集中在O-乙酰基酯与单体唾液酸的相互加成(NEUD)或减去(NEUA*)中所涉及的酶，在单体唾液酸与聚唾液酸结合之前。2)用创新的流式细胞仪技术和显微方法来区分不同宿主间的乙酰化和非乙酰化相，以确定胶囊聚唾液酸在体内的修饰动力学。3)通过(I)构建所有区域1输出基因的框架缺失，(Ii)确定生物合成是否需要3-脱氧-D-甘露糖辛基葡萄糖，(Iii)鉴定与辅助蛋白KPSC相互作用的聚合酶结构域(S)，(Iv)利用量子点技术和K1特异性噬菌体确定胶囊输出的位置，以及(V)确定起始复合体的化学结构，确定连接多糖合成和输出的分子基础。 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，并与数十亿美元的医疗费用有关。抗生素耐药性的增加，加上缺乏安全有效的疫苗或其他药物疗法，支持了为新的治疗开发确定细菌靶点的研究工作。我们的最新应用集中在大肠杆菌K1多唾液酸壳多糖的乙酰化形式，存在于所有毒力最强的K1菌株中，以及在大多数ExPEC菌株中产生主要毒力因子的壳多糖生物合成的基本机制。