Characterization of a New Key Regulator for Phenazine Production in Pseudomonads

描述(由申请人提供)：在理解如何在致病细菌铜绿假单胞菌中转录调节已知毒力因子吩嗪的产生方面存在根本差距。这一缺口的持续存在是一个重要的问题，因为在填补这一缺口之前，很大程度上无法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类药物。为了解决这个问题，我们的长期目标是定义PA2449在全球生理学中的功能(S) 铜绿假单胞菌。具体地说，必须知道PA2449调控哪些基因和操纵子，以及PA2449对哪些信号做出反应才能激活转录。这项应用的总体目标将是确定PA2449在医学上相关的细菌菌株铜绿假单胞菌中的调节，并确定其在吩嗪类毒力因子产生中的作用。中心假设是PA2449编码一种增强子结合蛋白(EBP)转录因子，调节铜绿假单胞菌中吩嗪类药物的产生。这一假设是基于我们自己的初步发现，这些发现是通过比较野生型铜绿假单胞菌PAO1和铜绿假单胞菌PA2449转座子缺失菌株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PA2449调控的遗传网络的了解将产生新的策略，以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。在强劲的初步数据的指导下，假设将是 通过追求两个特定的目标进行测试：1)定义PA2449的调节子；2)定义PA2449的代谢谱。在第一个目标下，将使用申请人手中已被确定为可行的技术；A)进行微阵列转录研究以确定PA2449的调节子；以及B)进行染色质-免疫沉淀-外切核酸酶(CHIP-EXO)实验以确定PA2449在整个铜绿假单胞菌基因组中的DNA结合位点。在第二个目标下，研究人员将使用常见的高效液相色谱和LC-MS-MS方法来评估PA2449菌株的氨基酸利用情况。该方法具有创新性，因为它首次识别和表征了一种EBP，该EBP专门调节铜绿假单胞菌中吩嗪类毒力因子的产生。这项拟议的研究意义重大，因为它有望纵向推进和扩大对铜绿假单胞菌如何以及为什么产生吩嗪类药物的理解。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.