OXIDATIVE STRESS SYSTEMS IN PSUEDOMONAS AERUGINOSA

这项调查的主要目标是增加我们的 对铜绿假单胞菌致病机制的认识 慢性呼吸道感染的囊性纤维化患者。内 在这些患者的气道中，铜绿假单胞菌必须具有存活的机制 1）吞噬细胞介导的形式的大量氧化应激 氧自由基，2）该环境中的高P02，和3）绿脓菌素，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 应激时，铜绿假单胞菌产生超氧化物歧化酶（SOD， 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）和藻酸盐（细胞外 氧自由基）。 我们最近发现，海藻酸盐调节 复合物受到氧化应激的强烈刺激。 知之甚少 关于这些氧化应激及其对调节 藻酸盐生物合成途径，我们将进行彻底的搜索， 不再响应氧化还原循环诱导的胁迫的突变体 积极影响藻酸盐基因转录的试剂。 以下 获得的突变体的表征，我们将克隆一个调控 氧化还原（rdx）基因，其影响algD的转录， 藻酸盐生物合成途径中的关键酶。 表征 rdx基因将包括物理作图，DNA序列分析， 使用转录融合进行调节。 定义的突变体缺陷， 氧化还原应激调节因子将通过基因置换构建， 被描述。 探讨超氧化物歧化酶（SOD）的作用及其调节机制， 铜绿假单胞菌对氧化应激的整体反应，我们将构建 sodB突变体的基因置换，以确定Fe-SOD在 氧化应激和藻酸盐产生。 我们还将描述 并产生sodAsodB双突变体， 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 在铜绿假单胞菌的调控中，我们还将通过 使用基因互补策略。 如上所述，Kat突变体和Kat- 将构建转录融合体，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.