Characterization of Enhancer Binding Proteins in Pseudomonas aeruginosa

 描述（由申请人提供） 增强子结合蛋白（EBP）是一种转录因子，与铜绿假单胞菌的毒力相关，但在理解EBP的作用方面存在根本性的空白。这个缺口的持续存在是一个重要的问题，因为在它被填补之前，理解EBP如何以及为什么调节铜绿假单胞菌的基因表达在很大程度上是不可理解的。为了解决这个问题，我们的长期目标是确定几个新发现的EBP在铜绿假单胞菌的全球生理学中的功能。具体来说，必须知道哪些基因和操纵子受这些EBP的调节，以及EBP响应什么信号以激活转录。本申请的总体目标是确定医学相关细菌菌株铜绿假单胞菌中EBP的调节子，并确定它们在代谢和毒力因子产生中的作用。核心假设是EBP转录因子将负责调节铜绿假单胞菌中多种代谢和毒力因子的产生。该假设是基于我们自己的初步发现，这些发现是通过检查野生型铜绿假单胞菌PAO 1和我们实验室产生的铜绿假单胞菌EBP缺失菌株的生理和转录组学数据而产生的。拟议研究的基本原理是，EBP调控的遗传网络的知识将产生新的策略，以防止毒力因子的表达或抑制铜绿假单胞菌的一般代谢，从而限制其引起感染的能力。在强有力的初步数据的指导下，将通过追求两个具体目标来测试该假设：1）PA 1196的表征，PA 1196是一种被提出来调节PA 1195表达的蛋白质，其编码可以降解铜绿假单胞菌中的有毒甲基精氨酸的二甲基精氨酸二甲氨基水解酶;以及2）鉴定未表征的EBP的功能。在第一个目标下，将使用申请人手中已经确立为可行的技术，A）测量PA 1195基因的表达水平，该基因是推定的调节因子PA 1196的下游; B）通过EMSA评估PA 1196蛋白与PA 1196的5'调节区的结合;和C）。对PA 1197蛋白进行生物化学表征。在第二个目标下，我们将使用转录组学和删除策略来确定铜绿假单胞菌的其余五个未表征的EBP的功能。该方法是创新的，因为它识别和表征了铜绿假单胞菌（一种模式细菌病原体）的所有EBP。这项研究具有重要意义，因为它有望纵向推进和扩展对EBP调节铜绿假单胞菌代谢和毒力因子产生的理解。最终，这些知识有可能确定新的治疗靶点，以干扰铜绿假单胞菌中的毒力因子产生。

 DESCRIPTION (provided by applicant) There is a fundamental gap in understanding the role of transcription factors called enhancer binding proteins (EBPs) relate to virulence in the pathogenic bacterium, Pseudomonas aeruginosa. Continued existence of this gap represents an important problem because, until it is filled, understanding how and why EBPs regulate gene expression in P. aeruginosa will be largely incomprehensible. To address this, our long-term goal is to define the function(s) of several newly identified EBPs in the global physiology of P. aeruginosa. Specifically, it is imperative to know what genes and operons are regulated by these EBPs, and what signals the EBPs respond to in order to activate transcription. The overall objective of this application will e to define the regulons of EBPs in the medically relevant bacterial strain, P. aeruginosa, and determine their roles in metabolism and virulence factor production. The central hypothesis is that EBP transcription factors will be responsible for regulating a diverse set of metabolisms and virulence factor production in P. aeruginosa. The hypothesis is based on our own preliminary findings, which were generated by examining physiological and transcriptomic data for wild type P. aeruginosa PAO1 and a P. aeruginosa EBP deletion strains generated in our laboratory. The rationale for the proposed research is that knowledge of the genetic networks regulated by EBPs will generate new strategies to prevent the expression of virulence factors or to inhibit the general metabolism of P. aeruginosa and thereby restrict its ability to cause infection. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Characterization of PA1196, a protein proposed to regulate expression of PA1195 which encodes a dimethylarginine dimethylaminohydrolase that can degrade toxic methylarginines in P. aeruginosa and 2) Identifying the functions of uncharacterized EBPs. Under the first aim, techniques, which have been established as feasible in the applicant's hands, will be used A) to measure expression levels of the PA1195 gene which is downstream of the putative regulator, PA1196; B) evaluate the binding of the PA1196 protein to 5' regulatory regions of PA1196 by EMSA; and C). Biochemically characterize the PA1197 protein. Under the second aim we will use transcriptomics and deletion strategies to identify the functions of the five remaining uncharacterized EBPs of P. aeruginosa. The approach is innovative because it identifies and characterizes all of the EBPs of P. aeruginosa, a model bacterial pathogen. The proposed research is significant, because it is expected to vertically advance and expand understanding of EBPs regulate metabolism and virulence factor production in P. aeruginosa. Ultimately, such knowledge has the potential to identify new therapeutic targets to interfere with virulence factor production in P. aeruginosa.