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

摘要 好了！ 细菌利用c-di-GMP(c-di-GMP)作为二次信号分子传递环境信号。 表型变化，包括生物膜形成和毒力。C-di-GMP分两次降解 酶促步骤：1.磷酸二酯酶-A(PDE-A)线性化生成pGpG；2.将pGpG水解为 PDE-B的GMP。虽然对PDE-A酶进行了更广泛的研究，但我们最近才发现 寡核苷酸酶(Orn)作为铜绿假单胞菌的主要PDE-B。铜绿假单胞菌∆orn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，我们的初步调查 数据表明，∆orn突变体也不能在导管相关的小鼠模型中传播 尿路感染(CAUTI)。然而，潜在的分子机制仍然是个谜。兽人是 被描述为3‘到5’外切核糖核酸酶，被认为能从2-7个残基中裂解短寡核苷酸 在长度上，完成了RNA的降解。Orn在外切核酸酶中是独一无二的，因为它是唯一的 已知的在许多伽马变形杆菌中必不可少的成员。根据我们的数据，Orn似乎发挥了作用 在RNA降解和c-di-GMP信号之间的交叉点，但酶如何调节两者 人们对二核苷酸信号和全球RNA池知之甚少。要在这一基础知识之间架起桥梁 针对Orn的独特功能，我们制定了一个跨学科的研究计划。我们假设 Orn的酶和生理功能与流行的观点不同，即Orn的作用相当于 非特异性纳米核糖核酸酶。取而代之的是，基于与pGpG复合的Orn的新结构，揭示了 为二核苷酸底物优化的受限催化位点，我们认为Orn的主要功能是 二核苷酸的内切酶。进一步的初步数据表明，没有这种能力的生物 Encode orn有额外的基因作为PDE-B发挥作用。我们以前的出版物和初步数据 形成了支撑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信号与慢性感染。为了验证我们的假设，我们将完成以下工作 目的：1.表征PDE-BS识别和催化底物的分子基础；2.阐明 On底物偏好和对寡核苷酸池的影响；以及3.确定调控的途径 在慢性铜绿假单胞菌导管相关性尿路感染期间。结果来自我们的 拟议的研究将揭示PDE-B对二核苷酸的偏好的结构基础，提供 PDE-B是二核苷酸酶的生化证据揭示了PDE-B的丢失对 二核苷酸和寡核苷酸池的积累，并发现导致缺陷的调节变化 在慢性感染期间。这笔赠款的影响是理解RNA降解和 环二核苷酸信号转导，并为PDE-B指定一个与其一致的定义函数 观察了信号和感染中的生理作用。

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.