BORDETELLA CYCLASE--STRUCTURE AND BIOLOGICAL ACTIVITY

博德特氏菌环化酶--结构和生物活性

• 批准号: 2060613
• 负责人: ERIK L HEWLETT
• 金额: $ 25.31万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-09-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060613
• 关键词: Bordetella pertussis; adenylate cyclase; antibody formation; bacterial toxins; circular dichroism; clone cells; electron microscopy; enzyme mechanism; host organism interaction; human tissue; laboratory mouse; membrane transport proteins; monoclonal antibody; mutant; pertussis vaccine; protein purification; protein structure function; protein transport; virulence

Infection with Bordetella pertussis remains a cause of pediatric morbidity and mortality and adult morbidity worldwide. Adenylate cyclase (AC) toxin from B. pertussis is a major virulence factor hypothesized to be involved in evasion of host defenses and development of local pathology. In this project, AC toxin structure and function will be studied for three purposes: 1) understanding its mechanism of action; 2) considering it as a vaccine antigen in acellular pertussis vaccine; and 3) contributing to the overall knowledge of bacterial toxins which enter target cells (traverse intact cell membranes). AC toxin is a calmodulin-regulate protein which catalyzes the production of cyclic AMP. Its novel features include its ability to enter intact eukaryotic cells to produce supraphysiologic levels of cAMP and its ability to hemolyze erythrocytes. AC toxin is synthesized in an inactive form which requires modification by a separate protein (product of the cyaC gene) for expression of full toxin and hemolytic activities. The c-gene product (CyaC) will be expressed, isolated and characterized. Monoclonal antibodies will be prepared against CyaC to facilitate study of its site and mechanism of action. The activation of AC toxin by the product of CyaC will be studied in vitro, in order to determine site of protein modification and structural consequences. Activated AC toxin binds calcium and undergoes a conformational change which allows it to enter eukaryotic target cells. This conformational changes does not occur in toxin from mutants missing CyaC, despite the fact that the toxin from such strains binds calcium. The role of CyaC-dependent activation in the structural response to calcium will be explored. Using purified, activated AC toxin the binding and entry processes will be dissected using functional techniques as well as electron microscopy. The study of toxin structure and translocation across target cell membranes will be used to formulate a model of toxin entry which will be specific to AC toxin as well as generally applicable to toxins which cross host-cell membranes. The apparent production of a transmembrane pore by the toxin molecule will be investigated by electro-physiologic methods in intact cells and artificial membranes. Serum from children immunized with whole cell pertussis vaccines or convalescent from pertussis will be evaluated for antibody response to AC toxin. In addition, purified AC toxin will be tested for its capacity to protect mice from B. pertussis infection and death in the intracerebral and aerosol challenge models. Inactive mutants of AC toxin will be evaluated as genetically toxoided antigens if AC toxin protective activity is demonstrated. In summary, these structure/function studies will facilitate better understanding of the mechanism of AC toxin as well as bacterial toxin action in general and will provide an antigen potentially useful in the prevention of clinical pertussis.

百日咳波德氏菌感染仍然是小儿发病率的原因 全世界的死亡率和成人发病率。 腺苷酸环化酶（AC）毒素 来自百日咳B. b. b. b.是涉及的主要毒力因子 在逃避宿主防御和局部病理的发展方面。 在这个 项目，AC毒素的结构和功能将进行三个 目的：1）了解其作用机理； 2）将其视为 细胞百日咳疫苗中的疫苗抗原； 3）贡献 进入靶细胞的细菌毒素的总体知识 （遍历完整的细胞膜）。 AC毒素是钙调蛋白调节的 催化循环AMP产生的蛋白质。 它的新颖特征 包括其进入完整的真核细胞产生的能力 cAMP的上造型水平及其溶解红细胞的能力。 AC毒素以非活动形式合成，需要通过 单独的蛋白质（CYAC基因的产物）用于表达全毒素 和溶血活动。 C-Gene产品（CYAC）将被表达 孤立和特征。 单克隆抗体将准备针对 CYAC促进其部位和作用机理的研究。 这 通过CYAC的产物，将在体外研究AC毒素的激活 为了确定蛋白质修饰和结构的位点 结果。 活化的AC毒素结合钙并经历构象变化 这使其可以进入真核靶细胞。 这种构象 尽管事实 从这种菌株中的毒素结合钙。 CYAC依赖性的作用 将探索对钙的结构反应中的激活。 使用 纯化，激活的AC毒素结合过程和进入过程将是 使用功能技术以及电子显微镜解剖。 这 研究毒素结构和跨靶细胞膜的易位 将用于制定毒素输入模型 AC毒素以及通常适用于交叉宿主细胞的毒素 膜。 毒素明显产生跨膜孔 分子将通过完整的电生理方法研究 细胞和人造膜。 全细胞百日咳疫苗免疫的儿童血清或 百日咳疗养者将评估对AC的抗体反应 毒素。 此外，将测试纯化的AC毒素的能力 保护小鼠免受脑内的百日咳感染和死亡的侵害 气溶胶挑战模型。 将评估AC毒素的非活性突变体 如果AC毒素保护活性为遗传性毒素抗原 证明。 总而言之，这些结构/功能研究将有助于更好 了解AC毒素和细菌毒素的机制 一般行动，将提供一种潜在有用的抗原 预防临床百日咳。