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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：
7785345
负责人：
Eric Ross Vimr
金额：
$ 21.79万
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/7785345
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 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 immunogenic 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（由申请方提供）：肠外致病性大肠埃希菌（ExPEC）是主要的病原体，主要发生在非常年轻、年老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. coli O 18：K1表达O 18体细胞抗原和K1多聚唾液酸荚膜抗原，这两种抗原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，我们定义了唾液酸转运、合成、聚合、催化和修饰单体和多聚唾液酸的完整途径。修饰机制与一种新型细菌病毒（CUS-3）有关，该病毒携带可变相乙酰化酶基因，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靶向它们的方法用于新的治疗开发。我们设计了三个具体目标来检验该假设：1）通过遗传改变乙酰转移酶NeuD和乙酰酯酶NeuA*（NeuA-star）来确定单体唾液酸O-乙酰化对总体聚唾液酸修饰的贡献。该目的集中于在将O-乙酰基酯掺入聚唾液酸之前，参与O-乙酰基酯与单体唾液酸相互加成（NeuD）或相减（NeuA*）的酶。2)使用创新的流式细胞术技术和显微镜方法确定体内荚膜聚唾液酸修饰的动力学，以区分不同宿主隔室中的乙酰化和非乙酰化相。3)通过（i）构建所有区域1输出基因的框内缺失，（ii）确定生物合成是否需要3-脱氧-D-甘露-辛酮糖酸，（iii）鉴定与辅助蛋白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输出位点，和（v）确定所述引发复合物的化学结构。公共卫生关系：肠外致病性大肠杆菌（ExPEC）每年在美国造成数万人死亡，并与数十亿美元的医疗费用有关。抗生素耐药性的发生率不断增加，加上缺乏安全有效的疫苗或其他药物治疗，支持了针对新治疗开发的细菌靶点鉴定的研究工作。我们的更新申请集中在E.大肠杆菌K1多聚唾液酸荚膜多糖，存在于所有毒性最强的K1菌株中，并且是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。