GENETIC ANALYSIS OF CHOLERA TOXIN STRUCTURE AND FUNCTION

这个项目的长期目标是了解结构， 在分子水平上研究霍乱毒素的功能并应用这些知识 用于开发改良的霍乱疫苗。 于本 项目期间，我们将使用定点和区域诱变， 分析A和B多肽的结构-功能关系， 霍乱毒素（CT），分析霍乱弧菌的分泌机制 CTas和胞外蛋白，构建嵌合霍乱毒素 表达霍乱弧菌的另外的保护性抗原的新抗原， 粘膜免疫原性和佐剂性试验。 我们最近建造了 由碱性磷酸酶和CT的A2结构域组成的融合蛋白 并证明它可以在体内与天然CT-B组装， 功能性嵌合全毒素 这一发现为一部小说提供了基础。 构建嵌合霍乱毒素作为递送 外源抗原以高度免疫原性的形式附着于粘膜表面。 我们 CT-B的突变分析将表征所需的结构特征 用于形成B五聚体、装配全毒素和结合 神经节苷脂GM 1。 我们对CT-A的研究将分析其结构特征 所需的全毒素组装，催化活性和ADP刺激 核糖基化因子 我们产生的突变CT将被筛选， 先前表征的单克隆抗CT和抗LT抗体， 鉴定CT-A或CT-B中影响 构象依赖性表位。 我们最近克隆了一个基因（excA）， 是CT穿过V外膜转位所必需的。 胆汁。 我们将通过表征CT的分泌来分析其机制。 exc A基因产物并确定是否需要额外exc基因 用于排泄CT。 我们还将测试exc基因产物是否可以 作为霍乱的保护性免疫原。 CT是一种 已知有效的粘膜免疫原，它刺激粘膜免疫 对同时给予的其他抗原的反应。 分子 将通过比较粘膜的CT特征来分析这些特征的基础。 小鼠对野生型CT和缺陷型CT的免疫应答 特定功能（例如，CT-B的神经节苷脂GM 1结合活性或 CT-A的酶活性），并且嵌合CT新抗原将被测试用于 它们作为多价粘膜免疫原的效力。

The long range goals of this project are to understand the structure and function of cholera toxin at the molecular level and apply that knowledge for development of improved vaccines against cholera. During the current project period we will use site-directed and regional mutagenesis to analyze the structure-function relationships of the A and B polypeptides of cholera toxin (CT),analyze the mechanisms by which Vibrio cholerae excretes CT as and extracellular protein, and construct chimeric cholera toxin neoantigens that express additional protective antigens of V. cholerae to test for mucosal immunogenicity and adjuvanticity. We recently constructed a fusion protein consisting of alkaline phosphatase and the A2 domain of CT and demonstrated that it can assemble in vivo with native CT-B to form functional chimeric holotoxin. This finding provides the basis for a novel method to construct chimeric cholera toxins as vehicles for delivery of foreign antigens to mucosal surfaces in a highly immunogenic form. Our mutational analysis of CT-B will characterize structural features required for formation of B pentamers, assembly of holotoxin, and binding to ganglioside GM1. Our studies of CT-A will analyze structural features required for holotoxin assembly, catalytic activity and stimulation by ADP ribosylation factors. Mutant CTs that we generate will be screened with previously characterized monoclonal anti-CT and anti-LT antibodies to identify structural changes in CT-A or CT-B that affect expression of conformationally dependent epitopes. We recently cloned a gene (excA) that is required for translocation of CT across the outer membrane of V. cholerae. We will analyze the mechanism of CT excretion by characterizing the excA gene product and determining if additional exc genes are required for excretion of CT. We will also test whether exc gene products can function as protective immunogens against cholera. Ct is one of the most potent mucosal immunogens known, and it stimulates the mucosal immune response to other antigens administered simultaneously. The molecular basis for these properties of CT will be analyzed by comparing the mucosal immune responses of mice to wild type CT and mutant CTs deficient in specific functions (e.g., ganglioside GM1-binding activity of CT-B or enzymatic activity of CT-A), and chimeric CT neoantigens will be tested for their potency as multivalent mucosal immunogens.