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: 2066922
• 负责人: H ROBERT MASURE
• 金额: $ 10.71万
• 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/2066922
• 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.

百日咳杆菌是一种世界性的致病菌，可引起百日咳 儿童和成人的持续性支气管炎， 天然水库 即使这个国家有疫苗计划， 据报道，有5000例百日咳病例， 未报告的病例，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， 百日咳成分，强调需要制定一个有效的 亚组分疫苗。 为了使这一努力取得成功， 了解细菌在宿主体内的生存策略 环境至关重要。 B。百日咳已经发展出一种生存策略， 协同调节毒力因子的表达， 设计成便于附着，从而将细菌建立在 主机和破坏主机防御。 这些毒力因子， 对于亚组分疫苗，被组织成vir调节子， 单个基因位点，BVG。 vir调节子的协调调节， bvg产生毒性和无毒力表型（表型调节）， 这种细菌对环境信号的反应。 明确定义的媒体 成分控制这种表达的文化，而鲜为人知的是， 用于表型调节的体内刺激。 至少两个不同 已经确定了B在宿主中的小生境。百日咳，细胞外， 纤毛上皮和细胞内，在吞噬细胞内， 从而提高了对这些毒力因子的表型调节的兴趣。 本建议的重点首先涉及协调机制， B中vir调节子的调节。百日咳，作为一个范例， 调节电路控制细胞外 腺苷酸环化酶毒素（ACT），该调节子的一个成员， 毒力决定因子 我们认为，多个调控因素 参与这场巡回赛。 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。 第二，bvg的目标 ACT操纵子内的调节将由体内DNA 260的足迹法和缺失、插入和定点突变 bp AT富集区，我们已经鉴定为含有 调控 第三，该调节回路的体内活性将被调节。 评估，在这种情况下，使用细菌入侵和生存内 人巨噬细胞作为实验模型。 这将是第一次 细菌快速转换的动力学和时间曲线 毒力调节因子在各种体内环境中是可检测的。 最后，提出了一个研究纲要，将生物化学和 从遗传学上描述一种新发现的蛋白质（CyaC）的作用 绝对需要的侵入性，溶血性和可能免疫原性 ACT的属性。