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）：人类病原体百日咳博德特氏菌引起称为百日咳的呼吸道感染咳嗽（百日咳）。今天，该疾病仍然是主要的肺部疾病国内和国际的健康问题。以百日咳为主发生在幼儿身上，但它也可以攻击所有年龄段的人。我们的关于百日咳博德特氏菌如何调节毒力基因表达的知识仍然不完整。这项研究拨款旨在调查其作用两个先前未鉴定的百日咳博德特氏菌转录调节因子， Btr（博德特氏菌转录调节因子）和 Baf（Bvg 附件）因子），控制博德特氏菌的代谢和发病机制。这些研究应该扩大我们对 B. 百日咳。这可能会导致人们更好地理解该病原体的生理学，以鉴定新的毒力因素，并更全面地了解这种微生物如何导致疾病。研究的目标是： 1) 确定和分离百日咳博德特氏菌的基因，其转录受 Btr 控制。最近开发的 Btr 滴定测定法将用于鉴定 Btr- 来自百日咳博德特氏菌基因组质粒库的调节基因。 Btr- 调节基因将被表征，选定的基因将被已测序。 2) 确定baf表达是否受 BvgAS 双组分调控系统并定义顺式作用参与 baf 表达的元件。一、野生型中的baf表达和 bvg 百日咳博德特氏菌菌株在存在或不存在的情况下生长调节剂（例如 MgSO4）将通过 Northern 分析进行检查和S1核酸酶保护测定。二、5'-上游区域 baf的表达和调节将通过分析来确定百日咳博德特氏菌中的染色体 baf 启动子-lacZ 转录融合。 3) 确定 Baf、BvgA 和 BvgS 如何相互作用以及具有 ptx 和 cya 基因的启动子区域。为了实现这一目标； Baf、BvgA 和 BvgS 蛋白将被过表达和纯化。这些调节蛋白与启动子区域的相互作用将是通过凝胶阻滞和 DNase I 保护分析进行分析。 4) 至确定 Btr、Btr 调节基因和 Baf 在百日咳博德特氏菌的毒力。突变将被构建在这些基因随后将被引入到毒力的染色体中 B. 通过基因替换技术治疗百日咳。 Btr 调控基因将分析突变体的生长特征，并且 Baf 缺陷突变体，将分析毒力因子水平生产。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。