METAL REGULATION IN HOST COLONIZATION BY B BURGDORFERI

BURGDORFERI 寄主定殖中的金属调节

• 批准号: 2827236
• 负责人: FRANK C GHERARDINI
• 金额: $ 20.79万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2827236
• 关键词: Bacillus subtilis; Borrelia; DNA footprinting; bacterial genetics; bacterial proteins; enzyme induction /repression; expression cloning; gel mobility shift assay; gene expression; genetic regulation; host organism interaction; iron; manganese; metal metabolism; metalloenzyme; metalloproteins; oxidative stress; peroxidases; superoxide dismutase; zinc

DESCRIPTION (Adapted from the Applicant's Abstract): Unlike other bacterial pathogens which must overcome host iron restriction to establish a successful infection, Borrelia burgdorferi, the causative agent of Lyme disease, is able to bypass iron limitation within a host by minimizing or perhaps even eliminating the need for iron. They accomplish this by eliminating pathways that include important iron- containing proteins and substituting other trace metals in metalloproteins that are found in B. burgdorferi. As a result, they do not appear to regulate gene expression based upon intracellular levels of iron as is seen in other bacterial pathogens. Instead, B. burgdorferi appears to regulate gene expression by monitoring levels of other metals, such as manganese or zinc. To investigate the observed metal- dependent gene expression, the PI has identified and cloned a gene encoding a putative metal-dependent repressor protein (PerR) from B. burgdorferi and identified a target sequence using a mobility shift DNA- binding assay. This sequence is 91 bp upstream of the start codon of a putative 2 gene operon encoding a glutamate transporter (gltP) and a NADH peroxidase (npx), suggesting that PerR may be involved in regulating an oxidative stress response by B. burgdorferi. A PerR homolog identified from Bacillus subtilis mediates cellular responses to oxidative stress and metal starvation in that bacterium. To understand the role this regulatory protein plays in the survival response of B. burgdorferi and to identify other genes it regulated, the PI proposed to (1) characterize PerR and its putative target sequence using mobility shift DNA-binding, primer extension, DNase I footprinting, and methylation/uracil interference assays, (2) assess the role of PerR and Nox in the oxidative stress response in B. burgdorferi by examining the effects of O2-, peroxide, and metal starvation on the expression of Nox and (3) identify additional genes regulated by PerR.

描述(改编自申请人的摘要)：不同于其他 必须克服宿主铁限制的细菌病原体 建立了成功感染伯氏疏螺旋体的致病因素 莱姆病的制剂，能够绕过宿主体内的铁限制 通过最小化甚至消除对铁的需求。他们 通过消除包括重要铁的途径来实现这一点- 含有蛋白质和取代其他痕量金属 在伯氏杆菌中发现的金属蛋白。因此，他们确实这样做了 似乎不是基于细胞内水平来调节基因表达 就像在其他细菌病原体中看到的那样。相反，B.burgdorferi 似乎通过监测其他基因的水平来调节基因的表达 金属，如锰或锌。为了研究观察到的金属- 依赖于基因的表达，PI已经鉴定并克隆了一个基因 编码一种可能的金属依赖阻遏蛋白(PerR)。 Burgdorferi，并使用迁移率改变DNA鉴定了目标序列- 结合试验。该序列位于A基因起始密码子上游91bp。 编码谷氨酸转运蛋白(GltP)的2个基因操纵子 NADH过氧化物酶(Npx)，提示PERR可能参与了 伯氏杆菌对氧化应激反应的调控。A Perr 从枯草芽孢杆菌中鉴定的同系物介导细胞反应 这种细菌中的氧化应激和金属饥饿。至 了解这种调节蛋白在生存中所起的作用 伯氏杆菌的反应及其调控的其他基因 PI用于(1)表征PERR及其推定的靶序列 使用迁移率移动DNA结合，引物延伸，DNase I 足迹和甲基化/尿嘧啶干扰分析，(2)评估 Perr和NOx在伯氏杆菌氧化应激反应中的作用 通过检测O2-、过氧化氢和金属饥饿对细胞的影响 NOx的表达和(3)确定受PerR调控的其他基因。