Chemistry & Immunochemistry of Exosporium Carbohydrates

B的最外表面层的组分。炭疽孢子称为外孢壁，是第一个与宿主接触的，它们与宿主防御系统的相互作用可能对摄入的孢子的命运产生重要影响。外孢壁的主要成分是一种称为BclA的糖蛋白。它含有约70个PTG三肽重复序列，其中几个具有由一个独特的取代氨基糖和三个鼠李糖残基组成的四糖侧链。BclA在炭疽病发病机制中的作用尚未确定，但已发现其他含鼠李糖的糖缀合物具有多种生物学活性。 有效的生物活性，包括激活巨噬细胞和抑制中性粒细胞吞噬作用。 该建议的第一个具体目的是鉴定BclA蛋白质骨架中糖基化的精确位置，并确定各个位点的取代程度。由于BclA的氨基酸序列不适合制备糖肽片段，因此该任务变得复杂，并且将使用新的分子遗传学方法来定位糖基化位点。第二个具体目标是确定参与BclA的含鼠李糖四糖组装的基因。这些基因将通过系统地突变失活候选基因来鉴定，所述候选基因是基于它们在B中的假定分配而选择的。炭疽菌基因组或其与其他炭疽菌中已知基因的相似性 细菌种类将确定这些基因突变对孢子形态、活力、萌发、感染性和毒力的影响。突变对寡糖结构的影响将使用各种化学和物理方法进行研究，包括质谱法和NMR方法。第三个具体目标是制备和评价一种新型寡糖-蛋白缀合物疫苗，其中BclA的四糖侧链共价连接到B的重组保护性抗原。炭疽病疫苗产生的抗体应能促进吞噬作用，杀死炭疽孢子，并防止毒血症。在一组实验中， 使用氢氧化铝佐剂皮下注射。在其他实验中，将其与霍乱毒素佐剂一起鼻内给药，以引发粘膜免疫应答。在这两种情况下，将评估疫苗保护易感小鼠品系免受致命吸入性炭疽感染的能力。

Components of the outermost surface layer of the B. anthracis spore, called the exosporium, are first to come in contact with the host, and their interactions with host defenses are likely to have an important influence on the fate of ingested spores. A major component of the exosporium is a glycoprotein called BclA. It contains about 70 PTG tripeptide repeats, several of which possess tetrasaccharide side chains composed of a unique substituted amino sugar and three rhamnose residues. The role(s) of BclA in anthrax pathogenesis has not been established but other rhamnose-containing glycoeonjugates have been found to possess a variety of potent biological activities, including activation of macrophages and inhibition of neutrophil phagocytosis. The first specific aim of this proposal is to identify the precise positions in the protein backbone of BclA that are glycosylated and also to determine the extent of substitution at the various sites. This task is complicated by the fact that the amino acid sequence of BclA is not amenable to the preparation of glycopeptide fragments and novel molecular genetic approaches will be used to locate the glycosylation sites. The second specific aim is to identify the genes involved in the assembly of the rhamnose-containing tetrasaccharides of BclA. These genes will be identified by systematically mutationally inactivating candidate genes selected on the basis of either their putative assignment in the B. anthracis genome or their similarity to known genes in other bacterial species. The effects of mutating these genes on spore morphology, viability, germination, infectivity, and virulence will be determined. The effects of the mutations on oligosaccharide structure will be studied using a variety of chemical and physical methods, including mass spectrometry and NMR methods. The third specific aim is to prepare and evaluate a novel oligosaccharide-protein conjugate vaccine in which the tetrasaccharide side chains of BclA are covalently linked to recombinant protective antigen of B. anthracis. Antibodies elicited by the vaccine should promote the phagocytosis and killing of anthrax spores as well as protect against toxemia. In one group of experiments the vaccine will be administered to mice subcutaneously using an aluminum hydroxide adjuvant. In other experiments it will be administered intranasally with a cholera toxin adjuvant in order to elicit a mucosal immune response. In both cases the ability of the vaccines to protect a susceptible strain of mice from a lethal inhalational anthrax infection will be assessed.