Chemical Structure of Anthrax Spore Polysaccharide

描述(由申请人提供)：我们研究的目标是确定炭疽孢子最外表面成分--外孢子菌多糖(EPS)的化学结构。炭疽芽孢杆菌的孢子是这种高致命性炭疽病的病原体。炭疽孢子由几个不同的结构层组成，保护核心遗传物质。由于其独特的结构特性，炭疽孢子寿命长，对恶劣环境具有极强的抵抗力。一旦进入宿主，同样的结构允许炭疽孢子在宿主免疫监视下存活并萌发，导致杆菌快速复制和产生杀死宿主的毒素。尽管有这些事实，但我们对炭疽孢子成分的基本化学结构及其功能知之甚少。 EPS在炭疽孢子表面表达，形成一层毛发状的孢子覆盖物。从休眠的孢子到新转化的营养细菌，EPS在整个萌发阶段都保持完好。由于多糖通常是免疫原性差的高度稳定的生物聚合物，我们推测EPS在保护孢子存活和保护孢子免受免疫防御方面发挥着重要作用。EPS结构的阐明将提供对炭疽孢子生物学和更广泛的产孢子菌的重要基础了解。EPS结构将揭示有用的抗原，用于开发快速检测和有效销毁炭疽孢子和/或史无前例的孢子导向炭疽疫苗和治疗方法。 目的1.从炭疽杆菌孢子中提取胞外多糖。孢子将从减毒的炭疽杆菌菌株中制备。外孢子层将从孢子中提取出来。EPS将通过生化和酶法从外孢子菌蛋白中分离出来。EPS将通过凝胶过滤和/或离子交换层析进行纯化。 目的2.阐明EPS的化学结构。多维高分辨核磁共振谱、质谱学、生化或酶降解、化学衍生化、以及气相色谱或高效液相色谱相结合的方法将用于阐明EPS的化学结构。如果发现EPS是异质的，我们将尝试描述所有不同实体的结构。

DESCRIPTION (provided by applicant): The goal of our study is to determine the chemical structure of the outermost surface component of anthrax spores, exosporium polysaccharide (EPS). Spores of Bacillus anthracis are the causative agent of the highly lethal anthrax infection. The anthrax spore consists of several distinct structural layers, which protect the core genetic material. Because of their unique structural properties, anthrax spores are long-lived and extremely resistant to adverse environments. Once inside the host, the same structures allow anthrax spores to survive the host immune surveillance and to germinate, leading to rapidly replicating bacilli and production of toxins that kill the host. Despite these facts, we know surprisingly little about the basic chemical structures of anthrax spore constituents and their functions. EPS is expressed on the surface of anthrax spores, forming a hair-like cover of the spore. EPS remains intact throughout the entire germination stage from dormant spores to newly transformed vegetative bacteria. Because polysaccharides are typically highly stable biopolymers of poor immunogenicity, we hypothesize that EPS plays significant roles in protecting the survival of spores and in shielding spores from immune defense. The elucidation of the EPS structure will provide vital fundamental understanding of anthrax spore biology and more generally of sporulating microorganisms. The EPS structure will reveal useful antigens for the development of methods for rapid detection and efficient destruction of anthrax spores and/or unprecedented spore-directed anthrax vaccine and therapy. Aim 1. Isolation of EPS from B. anthracis spores. Spores will be prepared from attenuated, avirulent strains of B. anthracis. The exosporium layer will be extracted from the spores. EPS will be cleaved from exosporium proteins by biochemical and enzymatic methods. EPS will be purified by gel filtration and/or ion exchange chromatography. Aim 2. Elucidation of the chemical structure of EPS. A combination of multidimensional high-resolution NMR spectroscopy, mass spectrometry, biochemical or enzymatic degradation, chemical derivatization, and gas chromatography or HPLC will be employed in parallel to elucidate the chemical structure of EPS. If EPS is found to be heterogenous, we will attempt to characterize the structures of all distinct entities.