Characterization of Enhancer Binding Proteins in Pseudomonas aeruginosa

 描述(由申请人提供) 在理解被称为增强子结合蛋白(EBPs)的转录因子在铜绿假单胞菌中与毒力相关的作用方面存在着根本的差距。这一缺口的持续存在是一个重要的问题，因为在它被填补之前，了解EBPs如何以及为什么调控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EBPs在铜绿假单胞菌全球生理学中的功能(S)。具体地说，必须知道哪些基因和操纵子受到这些EBPs的调控，以及EBPs对什么信号做出反应以激活转录。这项应用的总体目标将确定在医学上相关的细菌株铜绿假单胞菌中EBPS的调节，并确定它们在新陈代谢和毒力因子产生中的作用。中心假设是EBP转录因子将负责调节铜绿假单胞菌中一系列不同的代谢和毒力因子的产生。这一假设是基于我们自己的初步发现，这些发现是通过检查我们实验室产生的野生型铜绿假单胞菌PAO1和一株缺失EBP的铜绿假单胞菌的生理和转录数据而产生的。这项拟议研究的基本原理是，对EBPS调控的遗传网络的了解将产生新的策略，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。在强大的初步数据的指导下，将通过追求两个特定目标来验证这一假说：1)PA1196的鉴定，PA1196是一种调节PA1195表达的蛋白质，它编码一种能够降解铜绿假单胞菌中有毒甲基精氨酸的二甲基精氨酸二甲氨基水解酶；2)鉴定未鉴定的EBPs的功能。在第一个目的下，申请人手中已被确定为可行的技术将被用于A)测量假定的调节子PA1196下游的PA1195基因的表达水平；B)评估PA1196蛋白与PA1196的5‘调控区的结合；以及C)。对PA1197蛋白进行生化特征分析。在第二个目标下，我们将使用转录和缺失策略来鉴定铜绿假单胞菌剩余的五个未鉴定的EBPs的功能。这种方法是创新的，因为它识别和表征了铜绿假单胞菌的所有EBPs，一种模式细菌病原体。这项拟议的研究具有重要意义，因为它有望纵向推进和扩大对EBPS调节铜绿假单胞菌代谢和毒力因子产生的了解。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毒力因子的产生。

 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.