BORDETELLA CYCLASE-STRUCTURE AND BIOLOGICAL ACTIVITIES

描述(摘自申请者摘要)：腺苷环化酶毒素 (ACT)是波尔德氏菌产生的一种迷人的细菌毒力因子 百日咳。它具有不同寻常的特点，这使它成为 研究几种毒素家族的作用。ACT是一种1706种氨基酸 具有腺化环化酶结构域的蛋白质，负责催化 CAMP被输送到靶细胞胞浆后的产生 手机。此外，该分子也是细菌rtx家族的一员。 毒素和蛋白质，以其钙结合重复序列为特征。许多. 该家族的成员是形成孔洞的溶血素和细胞毒素，ACT也是 溶血，显然是通过在宿主细胞中形成一个低聚孔来实现的 薄膜。此外，这种毒素还能引起宿主细胞K+外流。 先于低聚物形成并可与低聚物形成分离的过程。完成工作 过去几年来，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作用的各个步骤以及如何 它们相互关联。 为了实现这一目标，本研究将遵循以下具体目标：1) ACT制备时的物理表征和对是否 任何或所有的活性都取决于二聚体的形成。 毒素；2)评估ACT与靶细胞的初始相互作用， 在任何膜植入之前，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被插入到 膜，使用电子顺磁共振(EPR)光谱，以及 确定该事件与K+流出的关系；4)映射 被传递到细胞质的催化结构域的一部分 中毒与K+外排/溶血是否相互作用的探讨 任何单一毒素分子的独家事件；以及5)表征 寡聚过程和所涉及的毒素结构域。 这项工作旨在了解这种毒素的作用机制。 一个非常基本的水平，有新的方法和技术。结果将是 确定这种毒素在百日咳发病机制中的重要作用 作为一项新的生物医学研究，更好地了解它的行为 将外来抗原运送到宿主免疫系统的探测器和载体。 此外，从拟议研究中获得的信息将有助于 到一般的毒素研究领域，在这个领域中有许多例子 大的外源蛋白通过不完全的方式进入宿主细胞 了解各种机制。

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.