权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

环二 GMP 信号传导和慢性生物膜感染的寡核糖核酸酶调节

基本信息

批准号：
10410509
负责人：
VINCENT T LEE
金额：
$ 57.9万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10410509
关键词：
5&apos -exoribonuclease Active Sites Affect Bacteria Bacterial Infections Bacterial Physiology Biochemical Biological Assay Bypass Catalysis Catalytic Domain Catheters Cell Culture Techniques Cell physiology Chronic Complement Complex Crystallization Cues Data Defect Degradation Pathway Dinucleoside Phosphates Enzymes Excision Exoribonucleases Family Feedback Gammaproteobacteria Gene Expression Genes Genetic Genetic Transcription Goals Grant Hydrolysis Infection Interdisciplinary Study Kidney Knowledge Length Microbial Biofilms Molecular Nucleotides Oligonucleotides Oligoribonucleotides Operon Organism Pathway interactions Periodicity Phenotype Phosphodiesterase I Physiological Process Property Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa infection Publications RNA RNA Degradation Recycling Regulation Ribonucleases Role Second Messenger Systems Signal Transduction Signal Transduction Pathway Signaling Molecule Structure Structure-Activity Relationship Testing Time Type III Secretion System Pathway Up-Regulation Virulence X-Ray Crystallography base catheter associated UTI chronic infection detection method diguanylate cyclase endonuclease gene function human pathogen in vivo member mouse model mutant nano oligoribonuclease pathogenic bacteria phosphoric diester hydrolase preference pyochelin response tripolyphosphate

项目摘要

ABSTRACT ! 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 physiological roles in signaling and infection.

摘要