Chemistry & Immunochemistry of Exosporium Carbohydrates

炭疽芽胞最外层的成分称为外孢子体，是首先与宿主接触的成分，它们与宿主防御系统的相互作用可能对摄取的孢子的命运产生重要影响。外孢子菌的一个主要成分是一种名为BCLA的糖蛋白。它包含约70个PTG三肽重复序列，其中几个具有四糖侧链，由一个独特的取代氨基糖和三个鼠李糖残基组成。BCLA在炭疽病发病中的作用(S)尚未确定，但已发现其他含鼠李糖的糖结合物具有多种 强大的生物活性，包括激活巨噬细胞和抑制中性粒细胞吞噬。 这项建议的第一个具体目标是确定BCLA蛋白骨架中糖基化的准确位置，并确定不同位置的取代程度。这项任务由于BCLA的氨基酸序列不适合糖肽片段的制备而变得复杂，将使用新的分子遗传学方法来定位糖基化位点。第二个特定的目标是确定与BCLA中含有鼠李糖的四糖组装有关的基因。这些基因将通过系统地突变失活候选基因来识别，这些候选基因是根据它们在炭疽菌基因组中的假定分配或它们与其他已知基因的相似性而选择的 细菌种类。突变这些基因对孢子形态、活力、萌发、侵染性和致病力的影响将被确定。这些突变对低聚糖结构的影响将使用各种化学和物理方法来研究，包括质谱学和核磁共振方法。第三个特异性目的是将BCLA的四糖侧链共价连接到炭疽杆菌的重组保护性抗原上，制备并评价一种新型的寡糖-蛋白质结合疫苗。疫苗产生的抗体应能促进炭疽孢子的吞噬和杀灭，并能预防毒血症。在一组实验中，疫苗将被注射到小鼠身上 皮下注射氢氧化铝佐剂。在其他实验中，它将与霍乱毒素佐剂一起鼻腔给药，以引起粘膜免疫反应。在这两种情况下，将评估疫苗保护敏感品系小鼠免受致命的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.