Characterization of a New Key Regulator for Phenazine Production in Pseudomonads

描述（由申请人提供）：在了解致病性细菌铜绿假单胞菌中吩嗪（已知毒力因子）的产生如何受到转录调控方面存在根本性差距。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，理解铜绿假单胞菌和其他假单胞菌如何以及为什么产生吩嗪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PA 2449在全球生理学中的功能， P.铜绿。具体而言，必须知道PA 2449调控哪些基因和操纵子，以及PA 2449响应哪些信号以激活转录。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PA 2449的调节子，并确定其在吩嗪类毒力因子产生中的作用。核心假设是PA 2449编码增强子结合蛋白（EBP）转录因子，该因子调节铜绿假单胞菌中吩嗪的产生。该假设是基于我们自己的初步发现，这些发现是通过比较野生型铜绿假单胞菌PAO 1和铜绿假单胞菌PA 2449转座子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，对PA 2449调控的遗传网络的了解将产生新的策略来防止这些毒力因子的表达，从而限制铜绿假单胞菌引起感染的能力，特别是在囊性纤维化肺中。在强有力的初步数据的指导下，假设将是 通过追求两个特定目标进行测试：1）定义PA 2449的调节子和2）定义PA 2449的代谢谱。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）进行微阵列转录组学研究以确定PA 2449的调节子;和B）进行染色质-免疫沉淀-核酸外切酶（ChIP-exo）实验以确定PA 2449在铜绿假单胞菌整个基因组中的DNA结合位点。在第二个目标下，研究人员将使用常见的HPLC和LC-MS-MS方法来评估PA 2449菌株的氨基酸利用率。该方法具有创新性，因为它首次鉴定和表征了特异性调节铜绿假单胞菌中吩嗪毒力因子产生的EBP。拟议的研究是重要的，因为它有望垂直推进和扩大对铜绿假单胞菌如何以及为什么产生吩嗪的理解。最终，这些知识有可能确定干扰铜绿假单胞菌毒力因子产生的新靶点。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how the production of phenazines, known virulence factors, is transcriptionally regulated in the pathogenic bacterium, Pseudomonas aeruginosa. Continued existence of this gap represents an important problem because, until it is filled, understanding how and why phenazines are produced by P. aeruginosa and other pseudomonads will be largely incomprehensible. To address this, our long-term goal is to define the function(s) of PA2449 in the global physiology of P. aeruginosa. Specifically, it is imperative to know what genes and operons are regulated by PA2449, and what signals PA2449 responds to in order to activate transcription. The overall objective of this application will be to define the regulon of PA2449 in the medically relevant bacterial strain, P. aeruginosa, and determine its roles in the production of the phenazines virulence factors. The central hypothesis is that PA2449 encodes an enhancer binding protein (EBP) transcription factor that regulates the production of phenazines in P. aeruginosa. The hypothesis is based on our own preliminary findings, which were generated by comparing physiological and transcriptomic data for wild type P. aeruginosa PAO1 and a P. aeruginosa PA2449 transposon deletion strain. The rationale for the proposed research is that knowledge of the genetic network regulated by PA2449 will generate new strategies to prevent the expression of these virulence factors and thereby restrict the ability of P. aeruginosa to cause infection, particularly in the cystic fibrosis lung. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Define the regulon of PA2449 and 2) Define the metabolic profile for PA2449. Under the first aim, techniques, which have been established as feasible in the applicant's hands, will be used; A) to perform microarray transcriptomic studies to define the regulon of PA2449; and B) to perform chromatin-immunoprecipitation-exonuclease (ChIP-exo) experiments to define the DNA binding sites of PA2449 throughout the genome of P. aeruginosa. Under the second aim, the investigators will use common HPLC and LC-MS-MS methodologies to assess the amino acid utilization of the PA2449 strain. The approach is innovative because it identifies and characterizes, for the first time, an EBP that specifically regulates the production of phenazines virulence factors in P. aeruginosa. The proposed research is significant, because it is expected to vertically advance and expand understanding of how and why phenazines are produced by P. aeruginosa. Ultimately, such knowledge has the potential to identify new targets to interfere with virulence factor production in P. aeruginosa.