OXIDATIVE STRESS SYSTEMS IN PSUEDOMONAS AERUGINOSA

这项调查的广泛目标是增加我们的 对铜绿假单胞菌致病机制的认识 囊性纤维化患者的慢性呼吸道感染。在 这些患者的呼吸道，铜绿假单胞菌必须有生存机制 吞噬细胞介导的相当大的氧化应激 氧自由基，2)该环境中的高P02，以及3)绿色素，a 铜绿假单胞菌产生的有毒氧化还原化合物。为了对抗这种氧化 在压力下，铜绿假单胞菌会产生超氧化物歧化酶(SODS)， 不成比例的超氧化物形成过氧化氢)，过氧化氢酶( 破坏过氧化氢)和海藻酸盐(一种胞外清道夫 氧自由基)。我们最近证明了藻酸盐的调节作用 复合体受到氧化应激的强烈刺激。鲜为人知 关于这些氧化应激及其对调节 藻酸盐的生物合成途径，我们将对 不再对氧化还原循环诱导的应激反应的突变体 对藻酸盐基因转录有积极影响的试剂。跟随 对获得的突变体进行鉴定，我们将克隆一个调节型 影响编码藻D基因转录的氧化还原(RDX)基因 藻酸盐生物合成途径中的关键酶。特征描述 RDX基因将包括物理作图、DNA序列分析和 利用转录融合进行调控。有缺陷的定义突变体 氧化还原应激调节子将通过基因替换构建，并将 被刻画出来。探讨超氧化物歧化酶的作用及其调控机制 铜绿假单胞菌对氧化胁迫的全球反应，我们将构建 通过基因置换确定Fe-SOD在SoDB突变体中的作用 氧化应激和藻酸盐的产生。我们还将描述 铜绿假单胞菌的苏打基因，并产生SodAsoDB双突变体 测定其对氧化应激的敏感性及对藻酸盐的影响 制作。确定过氧化氢酶的作用及其氧化剂的介导 在铜绿假单胞菌中的调控，我们还将通过以下方式克隆一个过氧化氢酶(Kat)基因 使用遗传互补策略。如上，凯特变种人和凯特- 将构建转录融合以评估 过氧化氢酶对各种氧化应激条件的反应。

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.