Biosynthesis of Polysaccharides

多糖的生物合成

• 批准号: 8633090
• 负责人: Peng George Wang
• 金额: $ 29.6万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-03 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8633090
• 关键词: Accounting; Adjuvant; Aftercare; Anabolism; Antibiotic Resistance; Biochemical; Biomedical Research; Cell Wall; Cell surface; Characteristics; Chemicals; Complex; Core Assembly; Cytoplasm; Diagnosis; Diphosphates; Distal; Drug Formulations; Endotoxins; Enzymes; Escherichia coli; Escherichia coli Infections; Evaluation; Event; Fluorine; Foundations; Funding; Gram-Negative Bacteria; Grant; Hospitalization; Human; Immune; Immunology; In Vitro; Individual; Infection; Investigation; Kidney Diseases; Length; Lipid A; Lipids; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Medical; Membrane; Modification; Mono-S; Morbidity - disease rate; Natural Immunity; Natural Resources; Nature; O Antigens; Oligosaccharides; Pathway interactions; Patients; Polymerase; Polymers; Polysaccharides; Prevalence; Prevention; Process; Proteins; Reaction; Recurrence; Research; Rest; Roentgen Rays; Side; Structure; TLR4 gene; Testing; Time; Toll-Like Receptor Pathway; Tube; Urinary tract infection; Urologic Diseases; Uropathogenic E. coli; Vaccine Adjuvant; Vaccine Design; Vaccines; Woman; analog; antimicrobial; base; drug candidate; glycosyltransferase; men; microbial; mortality; novel; novel vaccines; periplasm; polymerization; programs; public health relevance; reconstitution; resistance mechanism; resistant strain; standard care; sugar; tool; vaccine candidate

This proposed program is a competitive renewal of grant R01 GM085267 titled "Biosynthesis of Polysaccharides", funded from 7/30/2009 - 7/29/2013. This program is our continuing efforts to use both chemical and biochemical tools to elucidate the mechanism of polysaccharide biosynthesis and to produce promising drug candidates for biomedical evaluation. Lipopolysaccharides (LPS) are characteristic components of cell walls of Gram-negative bacteria, localize in the outer leaflet of asymmetric outer membrane (OM) and expose on the cell surface. LPS typically consists of a hydrophobic domain known as Lipid A (or endotoxin), a nonrepeating core oligosaccharide (including both the inner core and outer core) and a distal polysaccharide (O-antigen or O-PS). The biosynthetic pathway to LPS consists of independent biosynthesis of O-antigen and Core-Lipid A, respectively, and combination of these two parts to make LPS, and then transport to OM. Thus, total biosynthesis of LPS in vitro includes three Milestone events: 1) assembly of O-PS; 2) assembly of Core-Lipid A; 3) finally assembly of LPS. The first Milestone has already been completed in the last funding period of this grant. Biosynthesis of O-antigen of LPS is a wzy-dependent pathway: the individual repeating oligosaccharide unit is synthesized in the cytoplasm by the sequential action of specific glycosyltransferases. The repeating unit is then transported to the periplasmic side of the membrane by Wzx where it is polymerized into a polysaccharide by the polymerase Wzy. The chain length of the polymer is regulated by an unknown mechanism that involves Wzz protein. We used purified enzyme Wzy and Wzz to reconstitute such polymerization process, and for the first time, achieved the synthesis of polysaccharides in a test tube! Moreover, the enzyme WaaL (which transfers the polysaccharide from its diphosphate-lipid precursor to core-lipid A) was found to accept almost any structures of sugar-diphosphate-lipid donors. These results lay out an excellent foundation for accomplishing the remaining two milestones. For Milestone 2 of assembly of Core-lipid A, we will chemically synthesize a number of Lipid A molecules. The glycosyltranferases involved in the biosynthesis of core oligosaccharide will be over-expressed and used for in vitro sequential assembly of Core-Lipid A. The synthesized chemically defined Lipid A and Core-Lipid A molecules are not only useful research tools for studying LPS biosynthesis, but also potential vaccine adjuvants. Finally, Milestone 3 will be achieved by transferring the O-PS to the Core-Lipid A by WaaL to produce full LPS. Based on the understanding of Lipid A-TLR4/MD2 complex, many Lipid A molecules and Lipid A analogs have been found to have strong immune activities and can be used as adjuvant either alone or with other adjuvants in a variety of vaccine formulations. Thus, we hypothesize that our chemo-enzymatically constructed E. coli Inner Core-Lipid A and Core-Lipid A conjugates would represent as a novel set of wide- spectrum and stand-alone vaccine candidates with defined structures for prevention of urinary tract infection (UTI). Specifically, the program includes the following aims: Milestone 1: Our efforts in the biosynthesis of O-PS will include the synthesis of O-PS from two most commonly used uropathogenic E. coli (UPEC) strain CFT073 (O6:K2:H1) and UTI89 (O18:K1), and X-ray structure determination of WaaL and Wzy. Milestone 2: Chemically synthesize both E. coli di- and mono-phosphorylated lipid A with either tetraacylated or hexaacylated lipids, or with fluorine-containing hexaacylated lipid (total of 9 lipid A compounds), then transfer either E. coli R3 or R1 core oligosaccharides to these lipid A structures by following the biosynthetic pathway using sequential glycosyltransferase-catalzyed reactions. Milestone 3: Total assembly of E. coli O86 LPS, and other natural and chimeric LPS structures by WaaL catalyzed transformation. Successful execution of this research program should provide two unmet biomedical needs. Although LPS is one of widely used biochemicals in immunology and other biomedical research, there is essentially no pure LPS available. All the commercial LPS coming from isolation and purification from natural resources are inevitably a mixture. This program will achieve the total chemo-enzymatic synthesis of LPS, and open the field to investigate the structure-activity relation of LPS with its TLR4-MD2 complex. Moreover, the reconstituted, synthetic core-lipid A structures are novel wide-spectrum and stand-alone vaccine candidates against urinary tract infection caused by uropathogenic E. coli.

这项拟议的计划是R01 GM085267资助的竞争性续订，题为“生物合成 多糖“，从7/30/2009-7/29/2013资助。这个计划是我们继续努力使用这两个 化学和生化工具，以阐明多糖的生物合成和生产机制 有望用于生物医学评估的候选药物。 脂多糖是革兰氏阴性菌细胞壁的特有成分， 定位于非对称外膜(OM)的外叶，暴露在细胞表面。LP通常 由一个称为脂类A(或内毒素)的疏水结构域组成，它是一种非重复的核心低聚糖 (包括内核和外核)和远端多糖(O抗原或O-PS)。这个 内毒素的生物合成途径包括O-抗原和核心脂A的独立生物合成， 并将这两部分结合在一起制成脂多糖，然后输送到OM。因此，细菌内毒素的总生物合成 体外实验包括三个里程碑事件：1)O-PS的组装；2)Core-Lipid A的组装；3)最终的组装 LP。 在这笔赠款的最后一个资助期，第一个里程碑已经完成。生物合成 内毒素的O-抗原是一种依赖wzy的途径：单个重复的寡糖单位是在 通过特定糖基转移酶的顺序作用而形成的细胞质。然后将重复单元传送 通过Wzx进入膜的周质侧，在那里它被Wzx聚合成多糖 聚合酶Wzy。聚合物的链长受一种涉及WZZ的未知机制的调节 蛋白。我们使用纯化的酶Wzy和WZZ来改造这种聚合过程，并首次 时间长了，实现了在试管中合成多糖！此外，Waal酶(它能转移 从它的二磷酸类脂前体到核心类脂A的多糖)被发现可以接受几乎所有 糖-二磷酸-脂质供体的结构。这些结果为实现以下目标奠定了良好的基础 剩下的两个里程碑。 对于核心-脂类A组装的里程碑2，我们将化学合成一些脂类A 分子。参与核心低聚糖生物合成的糖基转移酶将过度表达 并用于核脂A的体外序列组装，合成的化学定义的脂A和 核心脂蛋白A分子不仅是研究内毒素生物合成的有用研究工具，而且具有潜在的应用前景 疫苗佐剂。最后，通过Waal将O-PS转移到Core-Lipid A来实现里程碑3 以产生完整的内毒素。 基于对Lipid A-TLR4/MD2复合体的了解，许多Lipid A分子和Lipid A 已发现类似物具有很强的免疫活性，可单独或与其他药物一起用作佐剂。 各种疫苗配方中的其他佐剂。因此，我们假设我们的化学-酶 构建的大肠杆菌内核-脂蛋白A和核心-脂蛋白A结合物可表示为一组新的广谱. 用于预防尿路感染的具有明确结构的光谱和独立候选疫苗 (UTI)。 具体地说，该计划包括以下目标： 里程碑1：我们在O-PS生物合成方面的努力将包括从两个最大的 常用的致尿系大肠杆菌(UPEC)CFT073(O6：K2：H1)和UTI89(O18：K1)，以及X-射线 Waal和Wzy的结构测定。 里程碑2：化学合成大肠杆菌二磷酸化和单磷酸化的类脂A 或六酰基脂，或与含氟的六酰基脂(总共9种类脂A化合物)，然后 通过遵循生物合成的方法将E.ColiR3或R1核心低聚糖转移到这些脂质A结构上 途径使用顺序的糖基转移酶-过氧化氢反应。 里程碑3：Waal对E.ColiO86LPS以及其他天然和嵌合的LPS结构进行了完全组装 催化转化。 这项研究计划的成功实施将提供两个未得到满足的生物医学需求。虽然 脂多糖是一种广泛应用于免疫学等生物医学研究的生物化学物质，基本上没有 提供纯脂多糖。所有商业内毒素都是从自然资源中分离提纯而来的 不可避免地，这是一种混合物。该方案将实现内毒素的全化学-酶法合成，开辟了这一领域 探讨脂多糖及其TLR4-MD2复合体的构效关系。而且，重组后的， 人工合成的核心类脂A结构是新型广谱和独立的尿路疫苗候选者 由致泌尿系统的大肠杆菌引起的呼吸道感染。