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.

该拟议计划是一个竞争性的更新赠款R 01 GM 085267题为“生物合成的 多糖”，资助时间为2009年7月30日至2013年7月29日。这一计划是我们不断努力使用这两个 化学和生物化学工具来阐明多糖生物合成的机制， 用于生物医学评价的有前途的候选药物。 脂多糖（LPS）是革兰氏阴性菌细胞壁的特征组分， 定位于不对称外膜（OM）的外叶并暴露于细胞表面。LPS通常 由称为脂质A（或内毒素）的疏水结构域组成， （包括内核和外核两者）和远端多糖（0-抗原或0-PS）。的 LPS的生物合成途径分别由O-抗原和核心脂质A的独立生物合成组成， 并将这两个部分结合，制备LPS，然后转运至OM。因此，LPS的总生物合成 体外包括三个里程碑事件：1）O-PS的组装; 2）核心-脂质A的组装; 3）最终组装 LPS。 第一个里程碑已经在本补助金的最后一个资助期内完成。生物合成 LPS的O-抗原是一个wzy依赖性途径：单个重复寡糖单元在LPS中合成。 通过特异性糖基转移酶的顺序作用而进入细胞质。然后将重复单元 通过Wzx连接到膜的周质侧，在那里它被Wzx聚合成多糖。 聚合酶Wzy。聚合物的链长是由一个未知的机制，涉及Wzz调节 蛋白我们使用纯化的酶Wzy和Wzz来重构这样的聚合过程， 时间，实现了在试管中合成多糖！此外，酶WaaL（转移 发现从其二磷酸-脂质前体到核心-脂质A）多糖接受几乎任何 糖-二磷酸-脂质供体的结构。这些成果为实现 剩下的两个里程碑。 对于核心-脂质A组装的里程碑2，我们将化学合成许多脂质A 分子。参与核心寡糖生物合成的糖基转移酶将过表达 并用于核心-脂质A的体外顺序组装。合成的化学成分确定的脂质A和 核心脂质A分子不仅是研究LPS生物合成的有用工具， 疫苗佐剂。最后，将通过WaaL将O-PS转移至核心脂质A来实现里程碑3 以产生完整的LPS。 基于对脂质A-TLR 4/MD 2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的免疫活性， 在各种疫苗制剂中的其它佐剂。因此，我们假设我们的化学-酶促反应 构建了E.大肠杆菌内核-脂质A和核-脂质A缀合物将代表一组新的宽- 用于预防尿路感染的具有确定结构的谱和独立候选疫苗 （UTI）。 具体而言，该方案包括以下目标： 里程碑1：我们在O-PS生物合成方面的努力将包括从两种最重要的生物合成O-PS。 常用的尿路致病性E.大肠杆菌（UPEC）菌株CFT 073（O 6：K2：H1）和UTI 89（O 18：K1），以及X射线 WaaL和Wzy结构测定。 里程碑2：化学合成E.大肠杆菌二和单磷酸化脂质A与四酰化 或六酰化脂质，或与含氟六酰化脂质（总共9种脂质A化合物），然后 转移E. coli R3或R1核心寡糖与这些脂质A结构的生物合成 途径使用连续糖基转移酶催化反应。 里程碑3：E. coli O 86 LPS，以及其他天然和嵌合LPS结构，通过WaaL 催化转化 这项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物化学品之一， 纯LPS可用。来自天然资源的分离和纯化的所有商业LPS都是 不可避免地是一个混合体。该方案将实现LPS的化学-酶法全合成， 探讨LPS与TLR 4-MD 2复合物的构效关系。此外，重组后的， 合成核心脂质A结构是新的广谱和独立的针对尿激酶的疫苗候选物， 尿路感染由尿路致病性E.杆菌