Structure and Function of the B. anthracis Exosporium

项目总结 炭疽芽孢杆菌是炭疽病的病原体，是一种被认为是严重威胁生物恐怖主义的高致病性细菌。这种细菌最近在美国被用作恐怖主义武器，暴露了对这一威胁作出更有效反应的必要性。炭疽杆菌被用作生物武器，主要是因为它能形成持久的孢子。这些孢子可以通过多种途径进入人体， 萌发，并以营养细胞的形式生长，这通常会导致宿主在几天内死亡。 炭疽杆菌的天然菌株对常见的抗生素敏感，可用于治疗炭疽病。 然而，大规模使用抗生素在后勤上具有挑战性，在医学上也是不负责任的。此外，抗药性菌株可能会被用于未来的攻击。目前的炭疽疫苗被证明是有问题的。因此，需要新的策略来应对炭疽威胁，这些策略可能需要详细了解哺乳动物免疫系统与最外层的 炭疽芽胞--外孢子菌。外孢子体是与寄主防御的主要相互作用部位，是抗体和破坏性酶的屏障，也是孢子抗原的来源。 此外，它也是众多检测设备的目标。外孢子体由准晶基底层和外部毛状绒毛组成。基底层含有大约20种结构蛋白和酶，NAP由孢子表面的优势抗原--胶原样糖蛋白BCLA形成。 最近的研究表明，外孢子菌蛋白在孢子致病中起着重要作用。这项建议的目标是使用遗传学、生物化学和免疫学方法来扩大我们对外孢子菌蛋白的理解，重点是它们在外孢子菌组装、孢子活力、萌发和生长中的关键作用。 致命性。具体地说，我们将：(1)研究外孢子菌蛋白质的合成、定位和功能，重点是那些具有特殊性质的蛋白质(例如，共价修饰的结构蛋白和发芽降解酶)。(2)阐明BCLA附着到基底层的机制，这似乎是通过 新的机制需要BCLA的定点蛋白水解性裂解，然后共价连接到特定的基底层蛋白。(3)研究基底层的组装，重点是蛋白质结合伙伴和共价交联和修饰基底层蛋白质的酶。(4)确定BCLA的寡糖结合部位。至少有两种不同的寡糖，包括一种含有诊断糖类的五糖，与BCLA的胶原样区域内的多个位置O-连接。我们的结果将使炭疽病新的预防和诊断程序的开发成为可能。此外，我们的研究将揭示蛋白质-蛋白质相互作用、大分子组装、蛋白质附着到细胞表面、细菌蛋白质的糖基化和磷酸化以及宿主-病原体相互作用的一般重要机制。

PROJECT SUMMARY Bacillus anthracis, the causative agent of anthrax, is a highly pathogenic bacterium considered to be a serious threat as an agent of bioterrorism. The recent use of this bacterium as a terrorist weapon in the United States exposed the need for a more effective response to this threat. B. anthracis is used as a biological weapon primarily because it forms durable spores. These spores can enter the body through multiple routes, germinate, and grow as vegetative cells, which usually results in the death of the host within several days. Natural strains of B. anthracis are sensitive to common antibiotics, which can be used to treat anthrax. However, large-scale use of antibiotics is logistically challenging and medically irresponsible. In addition, antibiotic-resistant strains could be used in future attacks. The current vaccine for anthrax has proven problematic. Thus, new strategies are needed to respond to the anthrax threat, and these are likely to require detailed knowledge of the interactions between the mammalian immune system and the outermost layer of the B. anthracis spore-the exosporium. The exosporium serves as the primary interactive site with host defenses, acts as a barrier to antibodies and destructive enzyme, and is the source of spore antigens. Additionally, it is the target of numerous detection devices. The exosporium is comprised of a paracrystalline basal layer and an external hair-like nap. The basal layer contains roughly 20 structural proteins and enzymes, and the nap is formed by the collagen-like glycoprotein BclA, the dominant antigen on the spore surface. Recent studies suggest that exosporium proteins play an important role in spore pathogenicity. The goal of this proposal is to use genetic, biochemical, and immunological methods to expand our understanding of the exosporium proteins, focusing on their critical roles in exosporium assembly, spore viability, germination, and virulence. Specifically, we will: (1) Examine the synthesis, location, and function of exosporium proteins, focusing on those with special properties (e.g., covalently modified structural proteins and germinant-degrading enzymes). (2) Elucidate the mechanism of attachment of BclA to the basal layer, which appears to occur by a novel mechanism requiring site-specific proteolytic cleavage of BclA followed by covalent attachment to a specific basal layer protein. (3) Examine the assembly of the basal layer, focusing on protein binding partners and on enzymes that covalently cross-link and modify basal layer proteins. (4) Identify the oligosaccharide attachment sites of BclA. At least two different oligosaccharides, including a pentasaccharide containing the diagnostic sugar anthrose, are O-linked to multiple sites within the collagen-like region of BclA. Our results will enable the development of new preventive and diagnostic procedures for anthrax. Additionally, our studies will reveal generally important mechanisms of protein-protein interactions, macromolecular assembly, protein attachment to cell surfaces, glycosylation and phosphorylation of bacterial proteins, and host-pathogen interactions.