BORDETELLA PERTUSSIS VIRULENCE FACTOR GENE REGULATION

百日咳博德氏菌毒力因子基因调控

• 批准号: 2067882
• 负责人: NICHOLAS H CARBONETTI
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067882
• 关键词: Bordetella pertussis; Escherichia coli; bacterial genetics; fusion gene; gene complementation; gene mutation; genetic library; genetic regulation; genetic regulatory element; molecular cloning; transposon /insertion element; virulence

Bordetella pertussis, the causative agent of whooping cough, produces a number of factors which contribute to its pathogenic potential. Expression of the genes encoding these factors is under the overall control of the bvg locus, which encodes a two component signal transduction (response regulator) system consisting of an environmental sensor protein and a DNA- binding transcriptional activator protein. However, evidence suggests that the bvg system directly activates only some of the genes in the bvg regulon (such as bvg and fha), whereas others (such as ptx and cya) apparently require additional factors for their activation. Two mutant strains of B. pertussis have greatly decreased levels of transcription of the ptx and cya genes, while transcription of other genes of the bvg regulon is normal, a phenotype which suggests a defect in an additional factor specifically for ptx and cya promoter activation. This proposal will focus on the identification of such additional regulatory factors in the bvg regulon, particularly those which activate the ptx (and cya) promoters. Random and targeted transposon mutagenesis will be performed to obtain mutants with phenotypes suggesting defects in these regulatory factors. Cloning of the genes encoding ptx promoter activating factors from a library of B. pertussis DNA will be attempted by two methods: activation of a ptx promoter-reporter gene fusion in Escherichia coli and probing for ptx promoter sequence-specific binding proteins in a lambdalgt11 expression library. The mutant strains with decreased ptx and cya transcription will be analyzed further to identify the specific defect involved, and the wild type gene corresponding to the mutant gene in these strains will be cloned by complementation of the mutants. Also, the promoters of the other known genes of the bvg regulon will be analyzed to determine whether they are directly activated by bvg or require additional regulatory factors, and identification and cloning of these factors will be attempted using the same methods as for the ptx promoter activating factors. An understanding of the regulatory events involved in expression of virulence factors in B. pertussis will provide further insight into the infection and disease process of this pathogen and will be useful for development of improved vaccines to prevent the disease.

百日咳Bordetella budtussis是百日咳的病因，产生了 导致其致病潜力的因素数量。 表达 编码这些因素的基因在BVG的总体控制之下 位点，该基因座编码两个组件信号转导（响应 调节器）系统由环境传感器蛋白和DNA-组成 结合转录激活蛋白。 但是，有证据表明 BVG系统直接激活BVG调节中的某些基因 （例如BVG和FHA），而其他（例如PTX和CYA）显然 需要其他因素激活。 B的两个突变菌株。 百日咳大大降低了PTX和CYA的转录水平 基因，虽然BVG调节的其他基因的转录是正常的，但 表型暗示了专门针对其他因素的缺陷 PTX和CYA启动子激活。 该提案将重点介绍此类额外 BVG调节中的调节因素，尤其是激活的因素 PTX（和CYA）启动子。 随机和靶向的转座子诱变 将进行以表型的突变体进行表明缺陷的突变体 这些调节因素。 编码PTX启动子的基因克隆 B. trussis DNA库的激活因素将通过 两种方法：激活PTX启动子 - 重复蛋白基因融合 大肠杆菌和PTX启动子序列特异性结合的探测 lambdalgt11表达式库中的蛋白质。 突变菌株与 PTX和CYA转录的减少将进一步分析以识别 涉及的特定缺陷，野生型基因对应于 这些菌株中的突变基因将通过互补来克隆 突变体。 此外，BVG调节的其他已知基因的启动子 将分析以确定它们是由BVG直接激活还是 需要其他监管因素，以及识别和克隆 这些因素将使用与PTX相同的方法尝试 启动子激活因子。 对监管事件的理解 百日咳芽孢杆菌中参与毒力因子的表达将提供 进一步了解这种病原体的感染和疾病过程 将有助于开发改善疫苗以预防该疾病。