OXIDATIVE STRESS SYSTEMS IN PSUEDOMONAS AERUGINOSA

铜绿假单胞菌的氧化应激系统

• 批准号: 2066987
• 负责人: DANIEL J. HASSETT
• 金额: $ 10.3万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066987
• 关键词: Pseudomonas aeruginosa; alginates; catalase; cystic fibrosis; fusion gene; gene complementation; gene mutation; genetic mapping; genetic transcription; genetic transduction; molecular cloning; molecular pathology; nucleic acid sequence; opportunistic infections; oxidative stress; regulatory gene; superoxide dismutase

The broad objective of this investigation is to increase our understanding of a pathogenic mechanism of Pseudomonas aeruginosa in chronic respiratory infections of cystic fibrosis patients. within the airways of these patients, P. aeruginosa must have mechanisms to survive considerable oxidative stress in the forms of 1) phagocyte-mediated oxygen radicals, 2) high P02 within this milieu, and 3) pyocyanin, a toxic redox compound produced by P. aeruginosa. To combat this oxidative stress, P. aeruginosa produces superoxide dismutases (SODs, which disproportionate superoxide forming hydrogen peroxide), catalases (which destroy hydrogen peroxide), and alginate (a scavenger of extra-cellular oxygen radicals). We have recently shown that the alginate regulatory complex is strongly stimulated by oxidative stress. Little is known about these oxidative stresses and its contribution to the regulation of the alginate biosynthetic pathway, we will conduct a thorough search for mutants which no longer respond to stress induced by a redox-cycling agent that positively affects alginate gene transcription. Following characterization of the mutants obtained, we will clone a regulatory redox (rdx) gene which affects the transcription of algD, a gene encoding a key enzyme in the alginate biosynthetic pathway. Characterization of the rdx gene will include physical mapping, DNA sequence analysis, and regulation using transcription fusions. Defined mutants defective in the redox stress regulator will be constructed by gene replacement and will be characterized. To investigate the role of SOD and its regulation in the global response to oxidant stress in P. aeruginosa, we will construct sodB mutants by gene replacement to determine the role of Fe-SOD in oxidant stress and alginate production. We will also characterize the sodA gene of P. aeruginosa and generate an sodAsodB double mutant to determine its sensitivity to oxidative stress and effect on alginate production. To determine the role of catalase, and its oxidant-mediated regulation in P. aeruginosa, we will also clone a catalase (kat) gene by using genetic complementation strategies. As above, kat mutants and kat- transcriptional fusions will be constructed to evaluate the role of catalase in response to various conditions of oxidative stress.

这项调查的广泛目标是增加我们的 了解铜绿假单胞菌在 囊性纤维化患者的慢性呼吸道感染。在 这些患者的气道，铜绿假单胞菌必须具有生存的机制 1）吞噬细胞介导的形式的氧化应激很大 氧自由基，2）在此环境中的高p02和3）pyocyanin，a 铜绿假单胞菌产生的有毒氧化还原化合物。 对抗这种氧化 压力，铜绿假单胞菌产生超氧化物歧化酶（SODS， 过氧化氢酶（过氧化氢酶）的不成比例的超氧化物（它们 破坏过氧化氢）和藻酸盐（细胞外的清道夫 氧自由基）。 我们最近表明藻酸盐调节 氧化应激强烈刺激复合物。 鲜为人知 关于这些氧化应激及其对调节的贡献 藻酸盐生物合成途径，我们将彻底搜索 氧化还原循环引起的压力不再响应的突变体 积极影响藻酸基因转录的药物。 下列的 获得的突变体的表征，我们将克隆调节 氧化还原（RDX）基因影响ALGD的转录，ALGD的转录，一种编码的基因 藻酸盐生物合成途径中的关键酶。 表征 RDX基因将包括物理映射，DNA序列分析和 使用转录融合的调节。 定义的突变体有缺陷 氧化还原应力调节剂将通过基因置换构建，并将 被描述。 调查SOD的作用及其在 铜绿假单胞菌中对氧化应激的全球反应，我们将构建 通过基因替代品SODB突变体，以确定Fe-Sod在 氧化剂应激和藻酸盐产生。 我们还将表征 铜绿假单胞菌的苏打基因，并生成一个sodasodb双重突变体 确定其对氧化应激的敏感性和对藻酸盐的影响 生产。 确定过氧化氢酶的作用及其氧化剂介导的 在铜绿假单胞菌中调节，我们还将通过克隆过氧化氢酶（KAT）基因。 使用遗传互补策略。 如上所述，Kat突变体和Kat- 将构建转录融合以评估 过氧化氢酶响应各种氧化应激条件。