BIOSYNTHESIS OF ENTEROBACTERIAL COMMON ANTIGEN

这项研究的长期目标旨在阐明 细菌外膜的生物发生和组装机制 革兰氏阴性细菌。预计对这些知识的了解 原核生物中的过程将提供对一般情况的洞察 真核细胞膜生物发生和组装的机制 和细胞器。 这里提出的具体研究涉及确定 肠道细菌共同抗原(ECA)的生物合成机制。非洲经委会 是一种细胞表面抗原存在于所有 革兰氏阴性菌属于肠杆菌科。这个 Eca的血清学特异性由线性 N-乙酰-D-氨基葡萄糖杂多糖(G1cNAc)， N-乙酰-D-甘露糖醛酸(ManNAcUA)和4-乙酰氨基-4，6- 二脱氧-D-半乳糖(Fuc4NAc)。这些氨基糖形成了重复的 三糖单元大于4)-β-D-甘露糖-(1大于 4)-Alpha-D-Glcp-Nac-(1大于3)-D-Fucp4NAc-(1大于. 尽管这种分子的地方性分类学分布表明 唯一与革兰氏阴性肠杆菌相关的功能，没有 关于生物合成的功能或机制是已知的 这是分子。 我们在这里提出了一些实验，旨在定义 ECA的生物合成途径。这些实验将进行初步的 观察表明G1cNAc-焦磷十一烯酚 (G1cNAc-PP-Lid)是ECA合成的早期中间产物。因此， 我们将研究最初的三糖重复的可能性 通过将ManNAcUA和Fuc4NAc顺序转移到 来自核苷酸供体UDP-甘露糖和甘露糖的G1cNAc-PP-脂质 TDP-Fuc4NAc。后续的实验将针对 脂联型杂多糖的体外合成论证 包含多个重复单元的链，我们还将确定 链延长的机理。更多的实验将会受到关注 通过鉴定RFE突变体中ECA合成受阻的步骤 鼠伤寒沙门氏菌。 拟议的研究将利用分子生物学和 体内脉冲标记、体外多糖等生物化学 化合物的合成、分离、分离和结构表征 生物合成中间体。

The long term goals of this research are directed at elucidating the mechanisms involved in the biogenesis and assembly of the outer membrane in Gram-negative bacteria. It is anticipated that a knowledge of these processes in prokaryotic organisms will provide insights into general mechanisms involved in membrane biogenesis and assembly in eukaryotic cells and cell organelles. The specific research proposed here is concerned with determining the mechanism of biosynthesis of the enterobacterial common antigen (ECA). ECA is a cell surface antigen present in the outer membrane of all Gram-negative bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of N-acetyl-D-glucosamine (G1cNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-4,6- dideoxy-D-galactose (Fuc4NAc). These amino sugars form the repeating trisaccharide unit Greater than 4)-Beta-D-ManpNAcUA-(1 Greater than 4)-Alpha-D-Glcp-NAc-(1 Greater than 3)-D-Fucp4NAc-(1 Greater than. Although the parochial taxanomic distribution of this molecule suggests a function uniquely associated with Gram-negative enteric bacteria, nothing is known concerning either the function or mechanism of biosynthesis of theis molecule. We propose here experiments designed to define the initial steps in the pathway of ECA biosynthesis. These experiments will pursue preliminary observations which suggest that G1cNAc-pyrophosphorylundecaprenol (G1cNAc-PP-lipid) is an early intermediate in ECA synthesis. Accordingly, we will investigate the possibility that the initial trisaccharide repeat unit is synthesized by the sequential transfer of ManNAcUA and Fuc4NAc into G1cNAc-PP-lipid from the nucleotide sugars donors UDP-ManNAcUA and TDP-Fuc4NAc, respectively. Subsequent experiments will be directed at demonstrating the in vitro synthesis of lipid-linked heteropolysaccharide chains containing multiple repeat units, and we will also determine the mechanism of chain elongation. Additional experiments will be concerned with identifying the step at which ECA synthesis is blocked in rfe mutants of S. typhimurium. The proposed research will utilize techniques of molecular biology and biochemistry such as in vivo pulse-labeling, in vitro polysaccharide synthesis, and isolation, fractionation, and structural characterization of biosynthetic intermediates.