The role of the c-di-GMP specific phosphodiesterase NbdA in NO-induced biofilm dispersal in Pseudomonas aeruginosa

c-di-GMP 特异性磷酸二酯酶 NbdA 在 NO 诱导的铜绿假单胞菌生物膜分散中的作用

• 批准号: 314811096
• 负责人: Professorin Dr. Nicole Frankenberg-Dinkel
• 金额: --
• 依托单位: Fachgebiet Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/314811096?language=en
• 关键词: role; di; GMP; specific; phosphodiesterase

Dispersion is a process by which bacteria transit from a biofilm to a motile growth state to spawn novel communities in new locales. Alterations in c-di-GMP levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is one factor known to induce biofilm dispersion through stimulation of c-di-GMP degrading phosphodiesterase (PDE) activity. We have recently identified a membrane-anchored protein specifically involved in NO-induced biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa. NO-induced biofilm dispersion locus A is a multidomain protein consisting of a MHYT-GGDEF-EAL fusion. In addition to NbdA, several other proteins were shown to be involved in P. aeruginosa biofilm dispersal, including the chemotaxis transducer BdlA and the PDE DipA. In order to understand the mechanism of NO-induced biofilm dispersal we intend to verify the sensory function of NbdA using purified recombinant protein. Based on bioinformatic predictions the MHYT-domain is proposed to be a gas sensor domain functioning through bound copper ions. UV-Vis and inductively coupled plasma optical emission spectroscopy in combination with site-directed mutagenesis will be used to identify the nature of cofactor and the coordinating amino acid residues. In addition, PDE assays will be performed to establish whether NO has a stimulatory effect on PDE activity. As an nbdA knock-out mutant is unable to disperse upon NO treatment we will perform cross-complementation experiments in biofilm tube reactors using known dispersion loci in order to identify the order of signal transduction. In addition, an in vivo membrane-Strep-tagged protein interaction experiment (membrane-SPINE) will be used to find direct interaction partner(s) to NbdA. Previous data also suggested a transcriptional regulation as nbdA mRNA levels in NO-dispersed cells were elevated. Transcriptional regulation will be investigated employing LOV-based fluorescent reporter fusions and monitored using fluorescence microscopy in biofilms and NO-dispersed cells. Several transcriptional regulators known to be involved in NO-sensing will be tested for their contribution in transcriptional regulation. In this regard we will also test whether the source of NO (exogenous vs. endogenous) has an influence on dispersal. Here we will make use of a P. aeruginosa mutant strain that contains a structurally intact, but catalytically inactive nitrite reductase. As a tool for the SPP community and to study biofilm dispersal independent of chemical, potentially harmful signals (like NO) we will construct a light-controllable PDE based on the bacterial phytochrome of P. aeruginosa. As a long term goal we will investigate how the perceived signal is transduced into a cellular response using a combination of fluorescent reporter gene fusion and nucleotide capture compounds to identify missing links in the signal transduction pathway.

分散是细菌从生物膜过渡到能动生长状态以在新的场所产生新的群落的过程。c-di-GMP水平的改变已被证明与许多不同细菌中的生物膜分散有关。信号分子一氧化氮（NO）是已知通过刺激c-di-GMP降解磷酸二酯酶（PDE）活性来诱导生物膜分散的一种因子。我们最近发现了一个膜锚定蛋白，专门参与了一氧化氮诱导的生物膜扩散的机会致病菌铜绿假单胞菌。NO诱导的生物膜分散位点A是由MHYT-GGDEF-EAL融合体组成的多结构域蛋白。除了NbdA之外，还显示了几种其他蛋白质参与铜绿假单胞菌生物膜分散，包括趋化性换能器BdlA和PDE DipA。为了了解NO诱导的生物膜分散的机制，我们打算使用纯化的重组蛋白来验证NbdA的感觉功能。基于生物信息学的预测，MHYT域被认为是通过结合铜离子起作用的气体传感器域。将使用UV-Vis和电感耦合等离子体发射光谱法结合定点诱变来鉴定辅因子和配位氨基酸残基的性质。此外，将进行PDE测定以确定NO是否对PDE活性具有刺激作用。由于nbdA敲除突变体在NO处理后不能分散，我们将使用已知的分散位点在生物膜管反应器中进行交叉互补实验，以鉴定信号转导的顺序。此外，将使用体内膜-Strep-标记的蛋白质相互作用实验（膜-SPINE）来寻找NbdA的直接相互作用配偶体。以前的数据也表明了转录调节，因为在NO分散的细胞中nbdA mRNA水平升高。转录调控将采用基于LOV的荧光报告融合进行研究，并在生物膜和NO分散细胞中使用荧光显微镜进行监测。几个转录调控已知参与NO传感将测试其在转录调控的贡献。在这方面，我们还将测试是否NO的来源（外源性与内源性）有扩散的影响。在这里，我们将利用一个铜绿假单胞菌突变株，含有一个结构完整，但催化失活的亚硝酸盐还原酶。作为SPP社区的一种工具，并研究生物膜分散独立的化学，潜在的有害信号（如NO），我们将构建一个光可控PDE的基础上的细菌光敏色素的铜绿假单胞菌。作为一个长期的目标，我们将研究如何感知的信号被转导到一个细胞的反应，使用荧光报告基因融合和核苷酸捕获化合物的组合，以确定在信号转导途径中缺失的环节。