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.

该提议的计划是授予R01 GM085267的竞争性续签，标题为“生物合成 多糖”，从2009年7月30日至2013年7月29日。 化学和生化工具，以阐明多糖生物合成机理并产生 有前途的候选生物医学评估的药物。 脂多糖（LPS）是革兰氏阴性细菌细胞壁的特征成分， 位于不对称外膜（OM）的外部小叶中，并暴露在细胞表面。 LPS通常 由称为脂质A（或内毒素）的疏水结构域，一种非重复的核心寡糖 （包括内核和外核）和远端多糖（O-抗原或O-PS）。这 LPS的生物合成途径分别由O-抗原和核脂质A的独立生物合成组成 以及这两个部分的组合以制作LP，然后运输到OM。因此，LPS中LPS的总生物合成 体外包括三个里程碑事件：1）O-PS组装； 2）核脂质a的组装； 3）最后组装 LPS。 第一个里程碑已经在这笔赠款的最后一个资金期间完成。生物合成 LPS的O-抗原是WZY依赖性途径：在 特定糖基转移酶的顺序作用的细胞质。然后将重复单元运输 通过WZX到膜的周质侧，在该膜中将其聚合成多糖。 聚合酶Wzy。聚合物的链长受涉及WZZ的未知机制调节 蛋白质。我们使用纯化的酶Wzy和WZZ重建了这种聚合过程，并且是第一个 时间，实现了测试管中多糖的合成！此外，酶WAAL（转移 发现从其二磷酸脂质前体到核脂质a）的多糖几乎接受 糖 - 二磷酸脂供体的结构。这些结果为完成 剩下的两个里程碑。 对于核脂质A组装的里程碑2，我们将化学合成许多脂质A 分子。与核寡糖的生物合成有关的糖基转磷酸酶将过表达 并用于核脂质A的体外顺序组装A。合成化学定义的脂质A和 核脂质A分子不仅是研究LPS生物合成的有用研究工具，而且是潜在的 疫苗佐剂。最后，通过将O-PS转移到核脂质A中，将实现里程碑3 生产完整的唱片。 基于对脂质A-TLR4/MD2复合物的理解，许多脂质A分子和脂质A 已经发现类似物具有强大的免疫活性，可以单独或与之一起用作佐剂 其他各种疫苗配方中的佐剂。因此，我们假设我们的化学酶 构建的大肠杆菌内核脂质A和核脂A的结合物将表示为一组新颖的广泛 谱系和独立疫苗候选物具有定义的结构，可预防尿路感染 （UTI）。 具体而言，该计划包括以下目的： 里程碑1：我们在O-PS的生物合成方面的努力将包括最大两个的O-PS合成 常用的肝病大肠杆菌（UPEC）菌株CFT073（O6：K2：H1）和UTI89（O18：K1）和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的脂质A具有四酰化 或六酰化的脂质，或含氟六酰胺的脂质（总计9种脂质A化合物），然后 通过遵循生物合成，将大肠杆菌R3或R1核寡糖转移到这些脂质A结构 使用顺序糖基转移酶 - 催化反应的途径。 里程碑3：大肠杆菌O86 LPS的总组装，以及其他天然和嵌合LPS结构 催化转化。 该研究计划的成功执行应提供两个未满足的生物医学需求。虽然 LPS是免疫学和其他生物医学研究中广泛使用的生化物之一，基本上没有 纯LPS可用。来自自然资源的孤立和纯化的所有商业唱片都是 不可避免地是混合物。该程序将实现LPS的总化学酶合成，并打开场地 研究LPS与其TLR4-MD2复合物的结构活性关系。而且，重组， 合成核脂质A结构是新型广谱和独立疫苗候选尿液 尿道病大肠杆菌引起的道感染。