In vitro and Cellular Tools for Complex Polysaccharide Biosynthesis

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.