# Oligoribonuclease regulation of cyclic-di-GMP signaling and chronic biofilm infections

> **NIH NIH R01** · UNIV OF MARYLAND, COLLEGE PARK · 2020 · $576,646

## Abstract

ABSTRACT
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Bacteria use cyclic-di-GMP (c-di-GMP) as a secondary signaling molecule to relay environmental cues to
phenotypic changes, including biofilm formation and virulence. C-di-GMP is degraded in two sequential
enzymatic steps: 1. Linearization to pGpG by phosphodiesterase-A (PDE-A) and 2. Hydrolysis of pGpG to
GMP by PDE-B. While PDE-A enzymes have been studied more extensively, we only recently identified
oligoribonuclase (Orn) as the primary PDE-B in Pseudomonas aeruginosa. A P. aeruginosa ∆orn mutant
has elevated levels of pGpG and c-di-GMP, resulting in hyperbiofilm formation. Notably, our preliminary
data indicate that the ∆orn mutant is also unable to disseminate in a murine model of catheter-associated
urinary tract infection (CAUTI). However, the underlying molecular mechanisms remain enigmatic. Orn is
described as a 3' to 5' exoribonuclease that is thought to cleave short oligonucleotides from 2-7 residues
in length, completing RNA degradation. Orn is unique amongst exoribonucleases since it is the only
member known to be essential in many gammaproteobacteria. Based on our data, Orn appears to function
at the intersection between RNA degradation and c-di-GMP signaling, but how an enzyme regulates both
dinucleotide signaling and global RNA pools is poorly understood. To bridge this fundamental knowledge
gap regarding Orn's unique function, we developed an interdisciplinary research plan. We hypothesize
that Orn's enzymatic and physiological function deviates from the popular view that Orn acts as rather
unspecific nano-RNase. Instead, based on new structures of Orn in complex with pGpG that reveal a
constrained catalytic site optimized for dinucleotide substrates, we propose that Orn functions primarily as
an endonuclease for dinucleotides. Further preliminary data demonstrate that organisms that do not
encode orn have additional genes that function as PDE-B. Our previous publications and preliminary data
form the scientific premise underlying our overarching hypothesis that PDE-Bs are dinucleotidases that
regulate c-di-GMP signaling and chronic infection. To test our hypothesis, we will complete the following
aims: 1. Characterize the molecular basis for substrate recognition and catalysis by PDE-Bs; 2. Elucidate
Orn substrate preferences and effects on oligonucleotide pools; and 3. Determine the pathways regulated
by Orn during chronic P. aeruginosa catheter-associated urinary tract infections. Results from our
proposed studies will reveal the structural basis for PDE-Bs preference for dinucleotides, provide
biochemical evidence that PDE-Bs are dinucleotidases, reveal the impact of loss of PDE-Bs on the
accumulation of diucleotide and oligonucleotide pools, and uncover altered regulation leading to defects
during chronic infections. The impact of the grant is to understand the intersection of RNA degradation and
cyclic dinucleotide signaling and to assign a defined function for PDE-Bs that is consistent with their
observed physiol...

## Key facts

- **NIH application ID:** 9936148
- **Project number:** 5R01AI142400-02
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** VINCENT T LEE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $576,646
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9936148

## Citation

> US National Institutes of Health, RePORTER application 9936148, Oligoribonuclease regulation of cyclic-di-GMP signaling and chronic biofilm infections (5R01AI142400-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9936148. Licensed CC0.

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