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

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。 C-di-GMP 分两次连续降解 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) 作为铜绿假单胞菌中的主要 PDE-B。铜绿假单胞菌 Δorn 突变体 pGpG 和 c-di-GMP 水平升高，导致过度生物膜形成。值得注意的是，我们初步 数据表明 Δorn 突变体也无法在导管相关小鼠模型中传播 尿路感染（CAUTI）。然而，潜在的分子机制仍然是个谜。奥恩是 被描述为 3' 至 5' 核糖核酸外切酶，被认为可从 2-7 个残基中切割短寡核苷酸 长度，完成RNA降解。 Orn 在核糖核酸外切酶中是独一无二的，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，Orn 似乎起作用 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传导和全局 RNA 库知之甚少。架起这些基础知识的桥梁 针对奥恩独特功能的差距，我们制定了跨学科研究计划。我们假设 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 非特异性纳米RNA酶。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 二核苷酸的核酸内切酶。进一步的初步数据表明，不具有 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，我们将完成以下任务 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库的影响； 3. 确定调控途径 Orn 在慢性铜绿假单胞菌导管相关尿路感染期间。我们的结果 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节改变 慢性感染期间。这笔赠款的影响是了解 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 观察信号传导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.