BORDETELLA PERTUSSIS VIRULENCE FACTOR GENE REGULATION

百日咳杆菌是百日咳的病原体， 许多因素导致其致病潜力。 表达 编码这些因子的基因中的每一个都在BVG的总体控制之下 基因座，其编码双组分信号转导（响应 调节器）系统，其由环境传感器蛋白和DNA- 结合转录激活蛋白。 然而，有证据表明， BVG系统仅直接激活BVG调节子中的一些基因 (such如bvg和fha），而其他（如ptx和cya）显然 需要额外的因素来激活它们。 两株B. 百日咳使PTX和CyA的转录水平大大降低 基因，而bvg调节子的其他基因的转录是正常的， 表型，这表明在一个额外的因素，特别是缺陷 PTX和CyA启动子激活。 本提案将侧重于确定此类额外的 BVG调节子中的调节因子，特别是那些激活 PTX（和CyA）启动子。 随机和靶向转座子诱变 将进行，以获得表型提示缺陷的突变体， 这些监管因素。 ptx启动子基因的克隆 来自B文库的活化因子。百日咳DNA将尝试由 两种方法：激活PTX启动子-报告基因融合体， 大肠杆菌及其ptx启动子序列特异性结合的研究 在一个表达文库中的蛋白质。 突变株与 将进一步分析减少的PTX和CyA转录， 所涉及的特定缺陷，以及对应于所述缺陷的野生型基因， 这些菌株中的突变基因将通过互补 变种人 此外，bvg调节子的其他已知基因的启动子 将被分析以确定它们是由bvg直接激活还是 需要额外的调节因子，以及鉴定和克隆 将使用与ptx相同的方法来尝试这些因素 启动子激活因子。 对监管事件的理解 参与B中毒力因子的表达。百日咳可以提供 进一步了解这种病原体的感染和疾病过程， 将有助于开发预防该疾病的改良疫苗。

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.