ADENYLATE CYCLASE TOXIN FROM BORDETELLA PERTUSSIS

百日咳博德特氏菌腺苷酸环化酶毒素

• 批准号: 2066923
• 负责人: H ROBERT MASURE
• 金额: $ 11.62万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-12-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066923
• 关键词: Bordetella pertussis; DNA footprinting; adenylate cyclase; bacterial genetics; bacterial proteins; genetic library; genetic regulation; host organism interaction; human tissue; macrophage; molecular cloning; nucleic acid sequence; pertussis toxin; phenotype; site directed mutagenesis; transcription factor; transposon /insertion element; virulence

Bordetella pertussis, is a worldwide pathogen which causes whooping cough in children and persistent bronchitis in adults who probably serve as the natural reservoir. Even though there is a vaccine program in this country, there are 5000 reported cases of whooping cough and an estimated 50,000 unreported cases, an incidence of disease on par with bacterial meningitis. The high reactogenicity of the diphtheria-pertussis-tetanus vaccine, due to the pertussis component, underscores the need to develop an effective subcomponent vaccine. In order to succeed in this effort, a thorough understanding of the survival strategy of the bacteria within the host environment is of paramount importance. B. pertussis has developed a survival strategy that relies on the coordinate regulation of the expression of virulence factors specifically designed to facilitate attachment thus establishing the bacteria within the host and to thwart host defenses. These virulence factors, the candidates for subcomponent vaccines, are organized into the vir regulon controlled by a single genetic locus, bvg. Coordinate regulation of the vir regulon by bvg produces virulent and avirulent phenotypes (phenotypic modulation) of this bacteria in response to the environmental signals. Well defined media components control this expression in culture, while little is known about the in vivo stimulus for phenotypic modulation. At least two different niches in the host have been identified for B. pertussis, extracellular, on the ciliated epithelium and intracellular, within phagocytes, thus heightening interest in phenotypic modulation of these virulence factors. The focus of this proposal first addresses the mechanism of coordinate regulation of the vir regulon in B. pertussis, using as a paradigm the regulatory circuit controlling the expression of the extracellular adenylate cyclase toxin (ACT), a member of this regulon and a major virulence determinant. We propose that multiple regulatory elements participate in this circuit. First we will identify these elements by transposon mutagenesis and molecular cloning. Second, the target for bvg regulation within the ACT operon will be defined by in vivo DNA footprinting and deletion, insertion and site directed mutagenesis of a 260 bp AT rich region which we have identified as containing the site for regulation. Third, the in vivo activity of this regulatory circuit will be evaluated, in this instance using bacterial invasion and survival within human macrophages as an experimental model. This will be the first time that a kinetic and temporal profile of the rapid switching by a bacterial virulence regulator will be detectable in various in vivo environments. Finally, an outline of research is presented which will biochemically and genetically characterize the role of a newly identified protein (CyaC) absolutely required for the invasive, hemolytic and probably immunogenic properties of the ACT.

Bordetella buttussis是一种全球病原体，会引起百日咳 在儿童和持续的支气管炎中，成年人可能是 天然水库。 即使这个国家有疫苗计划， 有5000例百日咳病例，估计有50,000例 未报告的病例，与细菌性脑膜炎相当的疾病发生率。 白喉 - - 毛线毒素 - 触发疫苗的高反应生成性 百日咳组成部分强调了开发有效的需求 子分量疫苗。 为了在这项努力中取得成功，彻底 了解宿主中细菌的生存策略 环境至关重要。 B.百日咳已经制定了一种依赖的生存策略 特别针对毒力因子的表达调节 旨在促进附着，从而建立了细菌 主持人和阻止主机防御。 这些毒力因素，候选人 对于子组成疫苗，将组织到由 一个遗传基因座，BVG。 通过协调VIR规范的调节 BVG产生强烈而无毒的表型（表型调制）的 该细菌响应环境信号。 定义明确的媒体 组件控制着文化中的这种表达，而对 表型调节的体内刺激。 至少两个不同 已经确定了宿主中的hod中的壁ch 吞噬细胞内的纤毛上皮和细胞内，因此 对这些毒力因子的表型调节的兴趣增加。 该提案的重点首先解决了坐标的机制 百日咳芽孢杆菌中的Vir grogulon的调节，用作范式 控制细胞外表达的调节电路 腺苷酸环化酶毒素（ACT），该调节的成员和主要 毒力决定因素。 我们提出了多个监管元素 参加此电路。 首先，我们将通过 转座子诱变和分子克隆。 第二，BVG的目标 ACT操纵子内的调节将由体内DNA定义 足迹和删除，插入和位点指示260的诱变 富裕地区的BP，我们已经确定为包含的地点 规定。 第三，该调节电路的体内活性将是 在这种情况下，使用细菌侵袭和生存评估 人类巨噬细胞作为实验模型。 这将是第一次 通过细菌的快速切换的动力学和时间轮廓 毒力调节剂将在各种体内环境中被检测到。 最后，提出了研究的大纲，该研究将在生化上和 遗传表征了新鉴定的蛋白质（CYAC）的作用 侵入性，溶血性和可能免疫原性绝对需要 该法的属性。