In vitro and Cellular Tools for Complex Polysaccharide Biosynthesis

与细菌细胞表面相关的复杂聚糖在细菌在环境和宿主中的生存中起着核心作用。这些多糖可以抵抗环境和宿主的挑战，包括洗涤剂、宿主免疫过程以及渗透压和ph值的变化。从历史上看，很难将特定的毒力功能归因于单个多糖，并且存在一些相互矛盾的报道来描述这些关系。缺乏这方面知识的主要原因是在选择性检测和定量特定细菌多糖方面存在固有的困难，并且发现在聚糖生物合成途径中经常中断具有与关键的共同底物-磷酸bactoprenyl的隔离相关的复杂影响。这些问题既存在于细菌生物学中，也存在于在新物种中异种表达新聚糖的合成生物学方案中。本提案的主要目标是开发检测和量化磷酸bactoprenyl的工具，以帮助了解如何基于底物丰度优化聚糖表达，并开发互补工具来检测细菌表面和细胞裂解物中的聚糖。在本项目中，我们将重点研究三种关键聚糖：空肠弯曲杆菌n -连接寡糖和脆弱拟杆菌荚膜多糖a。具体目标一，我们将致力于开发一种化学酶固定化和细胞制备聚糖的系统，该系统可用于研究从抗体到凝集素的聚糖结合伙伴。空肠梭菌n链低聚糖的初步研究为我们在这一领域的成功提供了关键的背景。在第二个具体目标中，我们使用LC-MS中的新工具和目标1中开发的工具来研究磷酸bactoprenyl的丰度如何影响重组聚糖的生产。我们也采取了一种无偏倚的定向进化方法来改善大肠杆菌中重组聚糖的生产。利用这些信息，我们将构建一个更好地优化重组多糖表达的大肠杆菌菌株。该应用程序提供了一系列工具，微生物学家和分析化学家可以使用这些工具来研究糖科学中的关键系统，这些系统是新疗法的重要靶点。

Complex glycans associated with the cell surface of bacteria play central roles in bacterial survival in the environment and in hosts. Those glycans provide resistance to environmental and host challenges, including detergents, host immune processes, and changes in osmolarity and pH. Historically it has been difficult to ascribe specific virulence functions to individual polysaccharides, and several conflicting reports describing these relationships exist. The primary reason for this lack of knowledge is the inherent difficulty in selectively detecting and quantifying specific bacterial polysaccharides, and the discovery that often disruptions in glycan biosynthesis pathways have complex effects related to the sequestration of a key common substrate, bactoprenyl phosphate. These problems manifest in both the biology of bacteria, and in synthetic biology schemes to heterologously express new glycans in new species. The major goal of this proposal is to develop tools to detect and quantify bactoprenyl phosphate to help understand how to optimize glycan expression based on substrate abundance, and to develop complementary tools to detect glycans on the surface of bacteria and in cell lysates. In this program we will focus on three key glycans: Campylobacter jejuni N-linked oligosaccharide, and Bacteroides fragilis capsular polysaccharide A. In specific aim one we focus our efforts on developing a system for the immobilization of chemoenzymatically and cell prepared glycans that can be used to investigate glycan binding partners from antibodies to lectins. Preliminary work with the C. jejuni N-linked oligosaccharide provides key background on our success in this area. In the second specific aim we investigate how the abundance of bactoprenyl phosphate impacts production of a recombinant glycan using new tools in LC-MS and the tools developed in aim 1. We also take an unbiased directed evolution approach to improving recombinant glycan production in E. coli. Using this information we will build an E. coli strain better optimized for recombinant glycan expression. Together this application provides a series of tools that can be used by microbiologists and analytical chemists for the investigation of critical systems in glycoscience that are important targets for new therapeutics. Modified Specific Aims In vitro and Cellular Tools for Complex Polysaccharide Biosynthesis Bacterial surface polysaccharides play central roles in a wide range of biology and could serve as targets for novel anti-microbial agents, pathogen sensors, vaccine antigens, or other important therapeutics. Some bacterial surface polysaccharides, including the Bacteroides fragilis polymer capsular polysaccharide A (CPSA), a major focus of this proposal, could also serve as therapeutics themselves. These applications require robust methods to produce these materials that can be easily adapted from one type of polysaccharide to another. A majority of bacterial polysaccharides are produced in nature via similar pathways in which highly specific glycosyltransferases assemble them one sugar at a time appended to a membrane bound molecular anchor called bactoprenyl phosphate (BP). BP is a C55 isoprenoid that is key to the biosynthesis of peptidoglycan, lipopolysaccharides, capsules, exopolysaccharides, teichoic acids, and oligosaccharides linked to proteins. Our laboratory has established the development of fluorescent polyisoprenoids that serve as a robust scaffold for the enzymatic preparation of a variety of complex glycans from different pathogenic and symbiotic microbes. These reagents have been pivotal in assigning the roles of specific genes in oligosaccharide assembly systems and for characterizing the selectivity of the glycosyltransferases responsible for their production. While these reagents have been especially useful in vitro there have been limitations on using them with living cells. This is problematic because it is becoming increasingly appreciated that the complex interplay between glycan biosynthesis systems can have major impacts on the physiology of these organisms. However, current methods are limited by the specific detection of particular glycans on the surface of bacteria, and the detection of intermediate glycans formed when these biosynthetic systems are altered. Tools to deconstruct the interplay between glycan biosynthesis pathways and to selectively detect complex glycans on the surface of bacteria are desperately needed. Unlike the tools available in nucleic acid and protein biochemistry, few are readily available, adaptable, and general for glycoscience. Our fluorescently tagged BP has had limited applications in studying living cells because of the inability of bacteria to take up BP analogues and incorporate them into clear glycan production pathways. In addition, we have found that when we reconstruct foreign biosynthetic pathways into E. coli for recombinant glycan expression, the detection of the glycan on the surface of the bacteria can be problematic. These two problems are the major focus of this proposal. Here we will develop new tools for the investigation of glycan biosynthesis systems in cells, and for the detection of recombinant or native glycans produced by E. coli. We will then use these tools for the optimization of recombinant glycan expression to develop an E. coli strain for CPSA production. Together these systems continue to push towards the development of general tools available for glycoscience akin to those developed for nucleic acid and protein biochemistry. Specific Aim 1: Bacterial glycan immobilization and binding partner capture. In this aim we will take advantage of our expertise in the in vitro assembly of complex glycans on analogues of BP and genetic methods for producing cellular BP-linked glycans. Heptasaccharide from the pathogenic microbe Campylobacter jejuni and CPSA repeat units from the symbiotic B. fragilis will be immobilized onto magnetic beads. Immobilized glycans from these organisms will then be used to capture known interacting proteins as a model system for the capture of new glycan interacting probes. The methodology developed will be applicable to new systems for glycan interacting partner discovery as well as microarrays and other applications requiring selective glycan immobilization. Specific Aim 2: Impact of bactoprenyl phosphate abundance on recombinant CPSA glycan production. In this aim we test the hypothesis that bactoprenyl phosphate abundance has a major impact on recombinant glycan production. To do this we will prepare heavy atom labeled BP and BP-linked glycans to be used as internal standards in the LC-MS based quantification of isoprenoids in cell lysates. We will then test the impact of genetically modulating BP abundance on the production of recombinant CPSA and the impact on cell survival. Lastly, we will develop an unbiased analysis via directed evolution for additional genetic components that influence recombinant glycan production using CPSA expression as our model system in E. coli.