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.
The proposed research is relevant to public health because characterizing enhancer binding proteins will provide access to the regulatory mechanisms for metabolism and virulence factor production in Pseudomonas aeruginosa presenting new targets for therapeutic development. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge about the nature and behavior of living systems in order to enhance health, lengthen life, and reduce the burdens of illness.
