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

摘要 ! 细菌使用环二GMP（c-di-GMP）作为二级信号分子，将环境线索传递给 表型变化，包括生物膜形成和毒力。C-di-GMP以两种顺序降解 酶促步骤：1.通过磷酸二酯酶-A（PDE-A）线性化pGpG，2. pGpG水解成 PDE-B的GMP。虽然PDE-A酶已经被更广泛地研究，但我们最近才发现， 寡核糖核酸酶（Orn）作为铜绿假单胞菌中的主要PDE-B。铜绿假单胞菌突变体 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 数据表明，在导管相关性肿瘤的鼠模型中， 泌尿道感染（CITTI）。然而，潜在的分子机制仍然是谜。Orn是 被描述为3'至5'核糖核酸外切酶，被认为从2-7个残基切割短寡核苷酸 长度，完成RNA降解。ORN在核糖核酸外切酶中是独特的，因为它是唯一的 已知在许多γ-变形菌中是必需的成员。根据我们的数据，奥恩似乎 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 二核苷酸信号传导和全局RNA库的理解很少。为了将这些基本知识 关于奥恩的独特功能的差距，我们制定了一个跨学科的研究计划。我们假设 Orn的酶和生理功能偏离了流行的观点， 非特异性纳米RNA酶。相反，基于Orn与pGpG复合的新结构，揭示了一种新的 限制催化位点优化二核苷酸底物，我们建议Orn的功能主要是 二核苷酸的核酸内切酶。进一步的初步数据表明， 编码ORN具有作为PDE-B起作用另外的基因。我们以前的出版物和初步数据 形成了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。为了验证我们的假设，我们将完成以下操作 目标：1.表征PDE-Bs对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.确定调节的途径 在慢性铜绿假单胞菌导管相关尿路感染期间，源自我们的 提出的研究将揭示PDE-B对二核苷酸的偏好的结构基础， PDE-Bs是二核苷酸酶的生物化学证据，揭示了PDE-Bs的丢失对 二核苷酸和寡核苷酸池的积累，并揭示导致缺陷的调节改变 在慢性感染中。该基金的影响是了解RNA降解和 环二核苷酸信号传导并为PDE-B分配与其一致的定义功能 观察信号和感染的生理作用。

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.