Linear diribonucleotides regulation of bacterial physiology and chronic biofilm infections

Univ Of Maryland, College Park, College Park MD

Investigators

Abstract

ABSTRACT RNA degradation was thought to proceed through endonucleolytic fragmentation, followed by exoribonuclease trimming which generate short RNA fragments that are turned over into mononucleotides by oligoribonuclease (Orn). In the last funding period, we published data supporting that only specific enzymes (Orn, NrnA, NrnB, and NrnC) are able to cleave diribonucleotides into monoribonucleotides, and that prokaryotic organisms need to encode at least one diribonuclease to fulfill this specific function. These results support a new perspective on RNA degradation in which the short oligoribonucleotides are processed through a sequence of discrete steps involving distinct enzymes. In addition, linear diribonucleotides appear to be biologically active molecules since we report that mutants lacking these enzymes accumulate diribonucleotides and have altered cell growth, enhanced formation of biofilm, and sporulation defects. Here we present additional preliminary data supporting diribonucleotides as active signaling molecules in the cell including: 1. Specific enzymes generate diribonucleotides, 2. The only suppressor of Pseudomonas aeruginosa ∆orn is a cryptic diribonuclease, 3. Two enzymes in central metabolism are diribonucleotide- binding proteins, and 4. P. aeruginosa ∆orn has virulence defects in an animal model of catheter-associated urinary tract infection. Our past publications and preliminary data provide the scientific premise for our hypothesis that cells generate linear dinucleotides from RNA degradation and linearization of cyclic dinucleotides, which can bind target proteins to alter cell physiology and pathogenesis. To test these aims, we will perform the following specific aims: In Aim 1, we will characterize the generation and degradation of diribonucleotides by determining the structure of diribonucleases and triribonucleases in complex with their respective substrates and determine their biochemical functions. In Aim 2, we will identify effects of dinucleotides on bacteria’s metabolism and physiology by characterizing the binding proteins that specifically interact with linear diribonucleotides. Building on our success of identifying cellular diribonucleotide receptors, we will screen for additional proteins from open reading libraries of Pseudomonas aeruginosa and Bacillus antracis. We will exploit the strains available to us that lack all diguanylate cyclases to reveal whether the effect of linear diribonucleotides is independent of c-di-GMP signaling. In Aim 3, we will characterize the virulence defects of mutants lacking diribonucleases. We will employ existing mutants lacking diribonucleases, including P. aeruginosa ∆orn and. Using P. aeruginosa ∆orn, we will study the defects in chronic infection in a murine model of catheter-associated urinary tract infection. Results from these studies will advance our understanding of RNA degradation and open a new area of signaling by linear diribonucleotides with the potential to be applied to novel antibacterial strategies.