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：
10163122
负责人：
VINCENT T LEE
金额：
$ 57.78万
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/10163122
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 Cleaved cell 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.

抽象的！细菌使用环二 GMP (c-di-GMP) 作为辅助信号分子来传递环境信号表型变化，包括生物膜形成和毒力在两个连续的过程中被降解。酶促步骤： 1. 通过磷酸二酯酶-A (PDE-A) 线性化为 pGpG，2. 将 pGpG 水解为 PDE-B 的 GMP 虽然 PDE-A 酶已得到更广泛的研究，但我们最近才发现。寡核糖核酸酶 (Orn) 作为铜绿假单胞菌 Δorn 突变体中的主要 PDE-B。 pGpG 和 c-di-GMP 水平升高，导致超生物膜形成，值得注意的是，我们的初步结果是。数据表明，Δorn 突变体也无法在导管相关小鼠模型中传播然而，Orn 的潜在分子机制仍然是个谜。被描述为 3' 至 5' 核糖核酸外切酶，被认为可从 2-7 个残基切割短寡核苷酸就长度而言，完成 RNA 降解的 Orn 在核糖核酸外切酶中是独一无二的，因为它是唯一的。根据我们的数据，Orn 似乎在许多伽马变形菌中发挥重要作用。在 RNA 降解和 c-di-GMP 信号传导之间的交叉点，但酶如何调节两者人们对二核苷酸信号转导和全局 RNA 池的了解还很有限。针对奥恩独特功能的差距，我们制定了跨学科研究计划。奥恩的酶和生理功能偏离了流行的观点，即奥恩的作用更像是相反，基于 Orn 与 pGpG 复合物的新结构，揭示了针对二核苷酸底物优化的受限催化位点，我们建议 Orn 主要充当进一步的初步数据表明，生物体不具有二核苷酸的核酸内切酶。编码orn有额外的基因作为PDE-B，我们之前的出版物和初步数据。构成我们总体假设的科学前提，即 PDE-B 是二核苷酸酶，调节 c-di-GMP 信号传导和慢性感染为了检验我们的假设，我们将完成以下工作。目标： 1. 表征 PDE-B 底物识别和催化的分子基础； 2. 阐明 Orn 底物偏好和对寡核苷酸池的影响；以及 3. 确定调控的途径 Orn 在慢性铜绿假单胞菌导管相关尿路感染期间的结果。拟议的研究将揭示 PDE-B 对二核苷酸偏好的结构基础，提供 PDE-B 是二核苷酸酶的生化证据揭示了 PDE-B 缺失对二核苷酸和寡核苷酸池的积累，并揭示导致缺陷的调控这笔资助的影响是了解 RNA 降解和慢性感染之间的交叉点。环状二核苷酸信号传导，并为 PDE-B 分配与其一致的定义功能观察信号传导和感染中的生理作用。