铜绿假单胞菌氧化应激转录因子OxyR结构与功能的研究

The capability of organisms to alter their gene expression patterns in response to environmental changes is essential for their viability. Oxidative stress derived from reactive oxygen species (ROS) produced via normal aerobic metabolism or by antibiotics, or by stimulated human phagocytic cells during infections is one of the major challenges for those living organisms. Many bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Upon activation by elevated levels of ROS, OxyR rapidly changes its conformation and activates a large array of genes to defense against oxidative stress. Recently, using chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip), we identified 122 novel OxyR target genes, including all the previously identified defensive genes (ahpCF, ahpB, katA, and katB) and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), biofilm development (bdlA), and small non-coding RNAs (snRNAs). In order to better understand the molecular mechanisms underlying oxidative stress mediated by OxyR and expand our horizon on the structural basis of this important phenomenon, in this project, by using the human opportunistic pathogen Pseudomonas aeruginosa, we will: (1) unravel the functions of those newly identified OxyR regulon, such as snRNAs and biofilm development gene (bdlA). Specifically, we will use Northern Blotting analysis, gene mutagenesis technique and RNA-seq approaches to identify the expression pattern of those genes in response to oxidative stress and their corresponding regulons. Those will add new understanding of OxyR-mediated small RNA regulation in other bacteria in addition to E. coli. (2) solve the structure of OxyR-DNA complex by determining the conditions of crystal growth for OxyR-DNA complex and improving the quality of crystals obtained, thus finally grasping the picture of OxyR-DNA complex. The complete determination of this complex will surely provide the structural basis for oxidative stress response in bacteria since no information has ever been obtained. The complete understanding of structure-function of the redox-sensitive transcriptional regulator OxyR will reveal the structural basis of oxidative stress response in prokaryotes and might eventually lead to a therapeutic agent that would enhance the killing mediated by H2O2 during bacterial infections.

研究微生物抗氧化胁迫响应机制可以提供防治病原微生物的方法。细菌中氧化应激反应的调控主要由转录因子OxyR负责。该因子通过感受外界活性氧水平而被活化，之后迅速发生结构上的变化而具有DNA结合能力，最后通过激活与抗氧化胁迫相关基因的转录来应对氧化胁迫。申请者近期发现铜绿假单胞菌中OxyR能够在其基因组上结合至少122个位点。该结果不仅验证了之前鉴定的抗氧化胁迫相关基因，还鉴定出许多参与其他代谢途径的基因，如小分子非编码RNA（snRNA）。本项目拟在此基础上，以人体机会致病菌铜绿假单胞菌为研究对象，运用基因突变与RNA-seq技术，阐明OxyR调控的snRNA的生物学功能与snRNA的作用机制；运用生物化学与结构生物学技术，解析全长OxyR与DNA复合体的结构。项目的完成将加深人们对OxyR的生物学功能及细菌感受氧化胁迫结构机制的理解，同时为人们利用OxyR作为药物靶标防治细菌感染奠定了基础。

研究微生物抗氧化胁迫响应机制可以提供防治病原微生物的方法。细菌中氧化应激反应的调控主要由转录因子OxyR负责。该因子通过感受外界活性氧水平而被活化，之后迅速发生结构上的变化而具有DNA结合能力，最后通过激活与抗氧化胁迫相关基因的转录来应对氧化胁迫。申请者近期发现铜绿假单胞菌中OxyR能够在其基因组上结合至少122个位点。该结果不仅验证了之前鉴定的抗氧化胁迫相关基因，还鉴定出许多参与其他代谢途径的基因，如小分子非编码RNA（snRNA）。本项目在此研究基础之上，以人体机会致病菌铜绿假单胞菌为研究对象，运用基因突变与RNA-seq技术，进一步阐明了OxyR调控的snRNA具有全新的生物学功能以及OxyR调控参与的铁代谢转录因子PvdS在生物被膜中的新作用；此外，还对oxyR突变体的转录组进行了系统分析，发现了oxyR可能与QS调控相互关联，进一步建立了氧化胁迫与细菌毒力间的关系。最后，在前期研究基础之上，我们运用生物化学与结构生物学技术，试图解析全长OxyR与DNA复合体的结构，目前已经获得了分辨率良好的晶体，但是还需要进一步的优化才能获得最终结果。该项目的完成进一步加深了人们对OxyR调控的生物学功能及细菌感受氧化胁迫结构机制的理解，同时为人们利用OxyR作为药物靶标防治细菌感染奠定了基础。