BORDETELLA CYCLASE-STRUCTURE AND BIOLOGICAL ACTIVITIES

说明书（改编自申请人的摘要）：腺苷酸环化酶毒素 (ACT)是由博德特氏菌产生的一种迷人的细菌毒力因子 百日咳。它具有不寻常的功能，这使它成为一个重要的原型， 研究几种毒素的作用。ACT是1706个氨基酸 一种具有腺苷酸环化酶结构域的蛋白质，负责催化 cAMP被递送到靶细胞的细胞质后的产生 cell.此外，该分子是细菌RTX家族的成员。 毒素和蛋白质，其特征在于它们的钙结合重复序列。许多 该家族成员是成孔溶血素和细胞毒素，ACT也是 溶血性，显然是通过在宿主细胞中形成寡聚孔 膜的此外，这种毒素在一种细胞内可使K+从宿主细胞流出， 在低聚物形成之前并可与之分离的过程。加班 在过去的几年里，人们认识到ACT的这些活动是 分开的和不同的。作为回应，下一阶段的目标是 正在进行的项目是了解毒素行动的各个步骤，以及如何 它们彼此关联。 为了实现这一目标，本研究将遵循以下具体目标：1） ACT制备时的物理表征，以及是否 任何或所有的活性都依赖于二聚体的形成， 毒素; 2）评估ACT与靶细胞的初始相互作用， 在任何膜插入之前使用方法来定义早期步骤， 发生; 3）映射以了解毒素的哪一部分插入到 膜，使用电子顺磁共振（EPR）光谱，和 确定该事件与K+外排的关系; 4）绘制 递送至细胞质的催化结构域的一部分， 中毒和K+外排/溶血是否相互影响的研究 对于任何单个毒素分子的排他性事件;和5）表征 寡聚化的过程和所涉及的毒素的结构域。 这项工作旨在了解这种毒素的作用机制， a very basic基础level水平，with novel新approaches方法and technologies技术.结果将 对于确定这种毒素在百日咳发病机制中的作用很重要 以及更好地了解其作为一种新型生物医学研究的作用 用于将外源抗原递送至宿主免疫系统的探针和载体。 此外，从拟议研究中获得的信息将有助于 毒素研究的一般领域，其中有许多例子， 大的外源性蛋白质通过不完全的 了解机制。

DESCRIPTION (Adapted from the Applicant's Abstract): Adenylate cyclase toxin (ACT) is a fascinating bacterial virulence factor produced by Bordetella pertussis. It has unusual features, which make it an important prototype for study of the actions of several families of toxins. ACT is a 1706 amino acid protein that possesses an adenylate cyclase domain responsible for catalyzing the production of cAMP after it is delivered to the cytoplasm of the target cell. In addition, this molecule is a member of the RTX family of bacterial toxins and proteins, characterized by their calcium-binding repeats. Many of the family members are pore-forming hemolysins and cytotoxins and ACT is also hemolytic, apparently by formation of an oligomeric pore in the host cell membrane. In addition, this toxin elicits K+ efflux from host cells in a process that precedes and can be dissociated from oligomer formation. Work over the last several years has led to recognition that these activities of ACT are separate and distinct. In response, the objective of the next phase of this ongoing project is to understand the individual steps of toxin action and how they relate to one another. To accomplish this goal, the research will follow these Specific Aims: 1) physical characterization of ACT as it is prepared and evaluation of whether any or all of the activities are dependent on the formation of dimers by the toxin; 2) evaluation of the initial interaction of ACT with the target cell, using methods to define the early step before any membrane insertion is able to occur; 3) mapping to learn which portion of the toxin is inserted into the membrane, using electron paramagnetic resonance (EPR) spectroscopy, and determination of the relationship of that event to K+ efflux; 4) mapping of the portion of the catalytic domain which is delivered to the cytoplasm and investigation of whether intoxication and K+ efflux/hemolysis are mutually exclusive events for any single toxin molecule; and 5) characterization of the process of oligomerization and the domains of the toxin involved. This work is directed at understanding the mechanism of action of this toxin at a very basic level, with novel approaches and technologies. The results will be important for defining the role of this toxin in the pathogenesis of pertussis and for gaining better knowledge of its actions as a novel biomedical research probe and vehicle for delivery of foreign antigens to the host immune system. In addition, the information acquired from the proposed studies will contribute to the general field of toxin research, in which there are many examples of large extrinsic proteins gaining access to host cells by incompletely understood mechanisms.