ACCESSORY CHOLERA ENTEROTOXIN, ACE, MECHANISM OF ACTION

辅助霍乱肠毒素，ACE，作用机制

• 批准号: 2517249
• 负责人: MICHELE TRUCKSIS
• 金额: $ 10.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2517249
• 关键词: SDS polyacrylamide gel electrophoresis; bacterial proteins; bactericidal immunity; cholera; cholera toxin; enterotoxins; gastrointestinal epithelium; gel mobility shift assay; ion transport; membrane channels; microorganism culture; mutant; protein purification; protein sequence; recombinant proteins; second messengers; virulence

DESCRIPTION (Adapted from applicant's abstract): Three toxins of V. cholerae that increase short circuit current in Ussing chambers have been identified. They include cholera toxin (CT), Zot (zonula occludens toxin) which acts by disrupting tight junctions, and Ace (accessory cholera enterotoxin), the subject of this proposal. The investigator has identified, cloned and sequenced the ace gene. Preliminary studies using crude toxin extracts in animal models indicated that Ace acts by increasing transcellular ion transport. Ace increased short- circuit current in Ussing chambers and caused fluid secretion in ligated rabbit ileal loops, characteristic of a classic enterotoxin. The predicted protein sequence of Ace shows striking similarity to the product of the cystic fibrosis bene. Based on activity and structural correlates, the investigator proposes to examine two hypotheses for the mechanism of action of Ace. The "second messenger" model is that Ace binds to a receptor on the epithelial cell membrane and activates a second messenger which increases chloride secretion via an endogenous channel. The "pore-forming" model is that Ace forms a new channel by inserting into the epithelial cell membrane. The specific aims are to: 1) compare native and recombinant Ace by purifying and characterizing both proteins: 2) determine the presence and protective function of an immune response to Ace: 3) study the effects of Ace on cellular function to distinguish between the "second messenger" and "pore- forming" hypotheses of Ace activity; 4) identify protein domains contributing to Ace activity; and 5) study the role of each of the V. cholerae toxins in virulence by constructing isogenic chromosomal mutants. The investigator will use bacterial and molecular genetic and cell physiology methods to examine the mechanism(s) of action of Ace on the gastrointestinal epithelial cell. The long term objectives of the proposal are to enhance understanding of the role of Ace in cholera pathogenesis, to identify possible new mechanisms of action of toxins, and to determine how the toxins of V. cholerae interact to cause disease.

描述（改编自申请人的摘要）：V的三种毒素。 霍乱会增加USSING CHAMBERS的短路电流 已确定。它们包括霍乱毒素（CT），ZOT（Zonula occludens 毒素）通过破坏紧密连接和ACE（配件）起作用 霍乱肠毒素），该提案的主题。 研究者已经确定，克隆并测序了ACE基因。 在动物模型中使用粗毒素提取物的初步研究表明 王牌通过增加跨细胞离子转运而起作用。王牌增加了 在使用室中的短路电流，并导致流体分泌 绑有兔回肠环，经典肠毒素的特征。 ACE的预测蛋白序列显示与 囊性纤维化有益的产物。基于活动和结构 相关，研究人员建议检查两个假设 王牌的作用机理。 “第二使者”模型是王牌 与上皮细胞膜上的受体结合并激活A 第二使者通过内源性增加氯化物的分泌 渠道。 “孔形成”模型是ACE通过 插入上皮细胞膜。 具体目的是：1）比较本地和重组王牌 纯化和表征这两种蛋白质：2）确定存在 和对ACE免疫反应的保护功能：3）研究 ACE对细胞功能的影响区分“第二 Messenger”和“孔形成” ACE活动的假设； 4）确定 有助于ACE活性的蛋白质结构域； 5）研究 通过构建同源性，毒力中的每一种霍乱毒素毒素 染色体突变体。研究者将使用细菌和分子 遗传和细胞生理方法检查机制的机制 ACE对胃肠道上皮细胞的作用。长期 该提案的目标是增强对 霍乱发病机理中的ACE，以确定可能的新机制 毒素的作用，并确定V.霍乱的毒素如何 相互作用以引起疾病。