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

• 批准号: 10620734
• 负责人: VINCENT T LEE
• 金额: $ 58.02万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620734
• 关键词: 5' -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; 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; 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

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 呢 细菌使用环状-DI-GMP（C-DI-GMP）作为次级信号分子，将环境线索传递到 表型变化，包括生物膜形成和病毒。 C-DI-GMP在两个顺序中降解 酶促步骤：1。通过磷酸二酯酶A（PDE-A）和2。PGPG水解为PGPG。 PDE-B的GMP。尽管PDE-A酶已经进行了更广泛的研究，但我们直到最近才确定 铜核酸酶（ORN）作为铜绿假单胞菌中的主要PDE-B。铜绿假单胞菌∆ORN突变体 PGPG和C-DI-GMP的水平升高，导致倍生形成。值得注意的是，我们的初步 数据表明∆ORN突变体也无法在导管相关的鼠模型中传播 尿路感染（CAUTI）。但是，潜在的分子机制仍然神秘。 Orn是 被描述为3'至5'的驱虫核酸酶，被认为可以从2-7个残差中清除短寡核苷酸 长度，完成RNA降解。 Orn在驱虫核酸酶中是独一无二的 已知成员在许多γ-细菌中都是必不可少的。根据我们的数据，ORN似乎起作用 在RNA降解与C-DI-GMP信号传导之间的交集，但酶如何调节两者 二核苷酸信号传导和全球RNA池的理解很少。桥接这一基本知识 关于ORN独特功能的差距，我们制定了一个跨学科研究计划。我们假设 Orn的酶促和身体功能与流行的观点偏离了Orn的行为 非特异性纳米RNase。相反，基于与PGPG复杂的ORN的新结构，该结构揭示了一个 针对二核苷酸底物优化的受限催化位点，我们提出ORN的作用主要是 用于二核苷酸的核酸内切酶。进一步的初步数据表明，没有 编码ORN具有其他功能为PDE-B的基因。我们以前的出版物和初步数据 形成我们总体假设的科学前提，即PDE-BS是二核替代酶 调节C-DI-GMP信号传导和慢性感染。为了检验我们的假设，我们将完成以下内容 目的：1。表征PDE-BS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；和3。确定受调节的途径 在慢性铜绿假单胞菌导管相关的尿路感染期间由ORN进行。我们的结果 拟议的研究将揭示PDE-BBS偏爱二核苷酸的结构基础，提供 PDE-BS是二核苷酸的生化证据，揭示了PDE-BS损失对 二核苷酸和寡核苷酸池的积累，并发现调节变化导致缺陷 在慢性感染期间。赠款的影响是了解RNA降解和 循环二核苷酸信号传导，并为PDE-BS分配符合其一致的PDE-BS 观察到信号和感染中的身体作用。

项目成果

NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro.

• DOI: 10.1093/nar/gkac1091
• 发表时间: 2022-11-28
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Weiss, Cordelia A.;Myers, Tanner M.;Wu, Chih Hao;Jenkins, Conor;Sondermann, Holger;Lee, Vincent T.;Winkler, Wade C.
• 通讯作者: Winkler, Wade C.

Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

• DOI: 10.1093/nar/gkad662
• 发表时间: 2023-10-13
• 期刊: Nucleic acids research
• 影响因子: 14.9