Structure and Function of the B. anthracis Exosporium

炭疽杆菌外孢子的结构和功能

• 批准号: 8077664
• 负责人: CHARLES LEE TURNBOUGH
• 金额: $ 43.74万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8077664
• 关键词: Animals; Anthrax Vaccines; Anthrax disease; Antibiotic Resistance; Antibiotics; Antibodies; Antigens; Bacillus anthracis; Bacillus anthracis spore; Bacteria; Bacterial Proteins; Biochemical; Biological; Bioterrorism; Cell surface; Cells; Cessation of life; Collagen; Detection; Development; Devices; Diagnostic; Diagnostic Procedure; Engineering; Enzymes; Future; Genetic; Germination; Glycoproteins; Goals; Growth; Host Defense; Human; Immune system; Infection; Knowledge; Link; Location; Methods; Napping; Oligosaccharides; Pathogenicity; Phosphorylation; Play; Post-Translational Protein Processing; Preventive; Process; Production; Property; Protein Binding; Proteins; Reproduction spores; Role; Route; Site; Soil; Source; Stratum Basale; Structural Protein; Structure; Surface; United States; Vaccination; Vaccines; Variant; Virulence; crosslink; glycosylation; hair-like nap; killings; macromolecular assembly; mutant; novel; pathogen; pathogenic bacteria; protein protein interaction; resistant strain; response; small molecule; sugar; weapons

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.

项目摘要 炭疽杆菌是炭疽病的病原体，是一种高致病性细菌，被认为是一种严重的生物恐怖主义威胁。美国最近将这种细菌用作恐怖主义武器，这表明需要对这一威胁作出更有效的反应。B。炭疽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， 萌发并作为营养细胞生长，这通常导致宿主在几天内死亡。 B的天然菌株。炭疽病对常用的抗生素敏感，抗生素可用来治疗炭疽病。 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，耐药菌株可用于未来的攻击。目前的炭疽疫苗已被证明存在问题。因此，需要新的策略来应对炭疽威胁，并且这些策略可能需要详细了解哺乳动物免疫系统与炭疽最外层之间的相互作用。 B。炭疽孢子-外孢壁。外孢壁作为与宿主防御的主要相互作用位点，作为抗体和破坏性酶的屏障，并且是孢子抗原的来源。 此外，它是许多检测设备的目标。外孢壁由次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，绒毛由胶原样糖蛋白BclA形成，BclA是孢子表面的主要抗原。 最近的研究表明，孢子外壁蛋白在孢子致病性中起着重要的作用。本提案的目标是使用遗传学、生物化学和免疫学方法来扩展我们对外孢壁蛋白的理解，重点关注它们在外孢壁组装、孢子存活力、萌发和发育中的关键作用。 毒性具体来说，我们将：（1）检查外孢壁蛋白的合成，位置和功能，重点是那些具有特殊性质的蛋白（例如，共价修饰的结构蛋白和萌发剂降解酶）。(2)阐明BclA附着于基底层的机制，这似乎是通过 需要位点特异性蛋白水解切割BclA，然后共价连接到特异性基底层蛋白的新机制。(3)检查组装的基础层，侧重于蛋白质结合伙伴和酶，共价交联和修改基层蛋白。(4)确定BclA的寡糖附着位点。至少两种不同的寡糖，包括含有诊断糖蒽的五糖，与BclA的胶原样区域内的多个位点O-连接。我们的研究结果将有助于开发新的炭疽预防和诊断程序。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、蛋白质附着于细胞表面、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。

项目成果

Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium.

• DOI: 10.1016/j.jsb.2014.02.018
• 发表时间: 2014-04
• 期刊: JOURNAL OF STRUCTURAL BIOLOGY
• 影响因子: 3
• 作者: Rodenburg, Cynthia M.;McPherson, Sylvia A.;Turnbough, Charles L., Jr.;Dokland, Terje
• 通讯作者: Dokland, Terje