NEW TRANSCRIPTIONAL REGULATORS OF BORDETELLA PERTUSSIS

百日咳博德特氏菌的新转录调控因子

基本信息

批准号：
2672463
负责人：
Richard Lee Friedman
金额：
$ 16.96万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-08-01 至 1999-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the applicant's abstract): The human pathogen Bordetella pertussis causes the respiratory infection know as whooping cough (pertussis). Today the disease still remains a major pulmonary health problem nationally and internationally. Pertussis predominately occurs in young children, yet it can also attack people of all ages. Our knowledge of how B. pertussis regulates virulence gene expression is still incomplete. This research grant proposes to investigate the role of two previously unidentified B. pertussis transcriptional regulators, Btr (Bordetella transcriptional regulator) and Baf (Bvg accessory factor), in the control of Bordetella metabolism and pathogenesis. These studies should expand our understanding of gene regulation in B. pertussis. It will likely lead to a better understanding of the physiology of this pathogen, to the identification of new virulence factors, and to a more complete understanding of how this microbe causes disease. The objectives of the research are: 1) To identify and isolate genes of B. pertussis whose transcription is controlled by Btr. A recently developed Btr titration assay will be used to identify Btr- regulated genes from a B. pertussis genomic plasmid library. Btr- regulated genes will be characterized and selected genes will be sequenced. 2) To determine whether baf expression is regulated by the BvgAS two-component regulatory system and to define the cis-acting elements involved in baf expression. First, baf expression in wildtype and bvg B. pertussis strains grown in the presence or absence of modulating agents (e.g., MgSO4) will be examined by Northern analysis and S1 nuclease protection assays. Second, the 5'-upstream region of baf expression and regulation will be identified by analysis of chromosomal baf promoter-lacZ transcriptional fusions in B. pertussis. 3) To determine how Baf, BvgA, and BvgS interact with each other and with the promoter regions of the ptx and cya genes. To accomplish this; Baf, BvgA, and BvgS proteins will be overexpressed and purified. Interactions of these regulatory proteins with promoter regions will be analyzed via gel retardation and DNase I protection analysis. 4) To determine the role Btr, Btr-regulated genes, and Baf play in the virulence of Bordetella pertussis. Mutations will be constructed in these genes which will then be introduced into the chromosome of virulent B. pertussis by gene replacement techniques. The Btr-regulated gene mutants will be analyzed for growth characteristics and, along with the Baf-deficient mutant, will be analyzed for levels of virulence factor production. The persistence and lethality of these mutants will be tested in the mouse intranasal infection model.

描述（改编自申请人的摘要）：人类病原体百日咳bordetella discoption呼吸道感染称为咳嗽（百日咳）。如今，这种疾病仍然是主要的肺部在国内和国际上的健康问题。百日咳主要发生在幼儿中，但也可以攻击各个年龄段的人。我们的知识对百日咳调节毒力基因表达是如何的知识仍然不完整。这项研究赠款建议调查该角色在两个先前未识别的百日咳杆菌转录调节剂中， BTR（Bordetella转录调节器）和BAF（BVG附件因素），控制Bordetella代谢和发病机理。这些研究应扩大我们对B中基因调节的理解。百日咳。这可能会导致更好地理解这种病原体的生理学，以鉴定新的毒力因素，以及对这种微生物如何引起的更全面了解疾病。研究的目标是：1）识别和百日咳芽孢杆菌的分离基因，其转录受BTR控制。最近开发的BTR滴定测定法将用于识别BTR- 来自百日咳基因组基因组质粒文库的调节基因。 btr- 调节基因将被表征，选定的基因将是测序。 2）确定BAF表达是否由 BVGAS两组人调节系统并定义顺式作用 BAF表达涉及的要素。首先，野生型中的BAF表达和BVG B.百日咳菌株在存在或不存在的情况下生长调节剂（例如MGSO4）将通过北方分析检查和S1核酸酶保护测定法。第二，5'上游区域 BAF表达和调节将通过分析来确定百日咳B。染色体BAF启动子-Lacz转录融合。 3）确定BAF，BVGA和BVG如何相互作用与PTX和CYA基因的启动子区域。实现这一目标； BAF，BVGA和BVGS蛋白将过表达和纯化。这些调节蛋白与启动子区域的相互作用将是通过凝胶延迟和DNase I保护分析分析。 4）到确定BTR，BTR调节的基因和BAF在百日草的毒力。突变将在这些基因然后将其引入毒物的染色体 B.通过基因替代技术百日咳。 BTR调节的基因将分析突变体的生长特征，并与 BAF缺陷突变体，将分析毒力因子的水平生产。这些突变体的持久性和致命性将是在小鼠鼻内感染模型中进行了测试。