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: 3456001
• 负责人: H ROBERT MASURE
• 金额: $ 11.14万
• 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/3456001
• 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.

百日咳博德特氏菌是一种引起百日咳的世界性病原体 儿童和成人持续性支气管炎可能是 天然水库。 尽管这个国家有疫苗计划， 已报告 5000 例百日咳病例，估计有 50,000 例 未报告的病例，其发病率与细菌性脑膜炎相当。 白喉-百日咳-破伤风疫苗的高反应原性是由于 百日咳部分，强调需要制定有效的 亚成分疫苗。 为了使这项工作取得成功，必须进行彻底的 了解宿主内细菌的生存策略 环境至关重要。 百日咳博德特氏菌已经发展出一种依赖于 毒力因子表达的协调调控 旨在促进附着，从而在细菌内建立 宿主并挫败宿主的防御。 这些毒力因素，候选人 对于子成分疫苗，被组织成由 vir 调节子控制的 单个遗传位点，bvg。 病毒调节子的协调调节 bvg 产生有毒和无毒表型（表型调节） 这种细菌对环境信号做出反应。 明确定义的媒体 成分在培养物中控制这种表达，但人们对此知之甚少 表型调节的体内刺激。 至少有两个不同的 宿主中的生态位已被确定为百日咳博德特氏菌，细胞外， 纤毛上皮和细胞内，吞噬细胞内，因此 提高了人们对这些毒力因子的表型调节的兴趣。 该提案的重点首先涉及协调机制 百日咳博德特氏菌病毒调节子的调节，使用作为范例 控制细胞外表达的调节电路 腺苷酸环化酶毒素 (ACT)，该调节子的成员，也是一个主要的 毒力决定因素。 我们建议多重监管要素 参加这个巡回赛。 首先我们将通过以下方式识别这些元素 转座子诱变和分子克隆。 二、bvg的目标 ACT 操纵子内的调控将由体内 DNA 定义 [第 260 章] bp AT 丰富区域，我们已确定该区域包含以下位点 规定。 第三，该调节回路的体内活性将是 评估，在这种情况下使用细菌入侵和存活 人类巨噬细胞作为实验模型。 这将是第一次 细菌快速切换的动力学和时间曲线 毒力调节剂可在各种体内环境中检测到。 最后，提出了研究纲要，该研究纲要将从生化和 从基因角度表征新发现的蛋白质 (CyaC) 的作用 绝对需要侵入性、溶血性和可能的​​免疫原性 ACT 的属性。