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（由申请人提供）：肠外致病性大肠杆菌（EXPEC）是大多数年轻，老年或免疫强化的人口中的主要病原体，每年导致数以万计的死亡人数，医疗保健成本数十亿美元。例如，主要的Expec亚型，大肠杆菌O18：K1，表达O18和K1多氨基酸胶囊抗原是两个主要毒力因子，这些因素是使这些菌株成为新生儿细菌脑膜炎的主要原因。使用微生物遗传学和碳水化合物分析的创新方法，我们定义了单体和多氨酸的唾液酸转运，合成，聚合，分解代谢和修饰的完整途径。修饰机制与携带相变的乙酰基酶基因基因催化随机O-乙酰化的新型细菌病毒（CUS-3）有关。我们假设了解这些细菌抗原的体内功能将有助于针对新的治疗发育的方法。我们设计了三个特定的目的来检验这一假设：1）确定单体唾液酸o-乙酰化对整体多氨酸修饰的贡献，通过遗传转移酶，neud和乙酰酯酶，neua*（NEUA-StAR）。该目的侧重于o-乙酰酯涉及的酶添加（neud）或减去（neua*），然后再掺入单体唾液酸，然后再掺入多氨酸中。 2）使用创新的流式细胞仪技术和微观方法来确定体内囊囊多氨酸的动力学，以区分不同宿主隔室中乙酰化和未乙酰化相的动力学。 3）确定通过（i）通过（i）在所有区域1导出基因中构建中构建偶联的多糖的分子基础，（ii）确定生物合成（III）是否需要使用量子量的量子蛋白质（s）kpscccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccscccccccccccccccccc的量度和量很大，否则是否需要3-脱氧-d-甘露二二二旋链蛋白（spcc） K1-特异性噬菌体以确定胶囊导出位点，（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）每年在美国造成数以万计的死亡，并与数十亿美元的医疗保健费用有关。抗生素耐药性的发生率的增加，加上缺乏安全有效的疫苗或其他药物疗法的研究支持了针对鉴定新治疗性发育的细菌靶标的研究工作。我们的更新应用集中于大肠杆菌K1多氨基酸胶囊多糖的乙酰化形式，存在于所有最毒的K1菌株中，以及囊囊多糖生物合成的基本机制，从而在大型的Expecec菌株中产生主要的毒力因子。