Cyclic-di-GMP signaling in Pseudomonas aeruginosa

描述（由申请方提供）：本项目旨在确定次级细胞内代谢产物环二鸟苷酸（c-di-GMP）刺激机会致病菌铜绿假单胞菌形成生物膜的机制。生物膜被定义为包裹在自我产生的胞外多糖（EPS）基质中的表面相关的多细胞群落，是具有潜在倾向（如囊性纤维化）的人类慢性铜绿假单胞菌感染的原因。生物膜感染是有问题的，因为它们对抗生素治疗有抗性，并且往往逃避免疫监视。具有高c-di-GMP的铜绿假单胞菌细胞形成厚的生物膜和独特的褶皱集落，并且具有降低的运动性。铜绿假单胞菌中c-di-GMP的主要生物膜相关效应是刺激Psl和Pel EPS产生。这部分是由于c-di-GMP对pel和psl基因表达的影响。C-di-GMP还刺激EPS生物合成酶的活性。这种效应可能是由于特定的二鸟苷酸环化酶在特定的亚细胞位点产生c-di-GMP。虽然已经建立了c-di-GMP活性的一般参数，但c-di-GMP作用的机制才刚刚开始探索。所提出的实验将测试转录因子FleQ响应于c-di-GMP与FleN协同调节转录的模型。具有纯化的蛋白质和pel启动子DNA的体外系统将是探索机制的实验的起点（目的1）。其他实验将表征来自铜绿假单胞菌的最具活性的二鸟苷酸环化酶WspR的催化性质。WspR是一种杂合反应调节剂-二鸟苷酸环化酶，其在被磷酸化激活时合成c-di-GMP。wspR突变体在EPS合成中有缺陷。WspR的组成型活性突变体形式将被特别靶向用于表征（目的2）。荧光蛋白标记的WspR在磷酸化时在细胞的细胞质中形成动态簇，因此具有活性。这表明WspR-P在离散的亚细胞位置产生c-di-GMP，并暗示细胞具有c-di-GMP作用的特异性WspR相关靶标。将评估已知c-di-GMP受体蛋白的亚细胞位置及其与WspR的可能相互作用。还将进行实验以鉴定可能指导WspR-P分布的细胞骨架蛋白。此外，荧光显微镜将用于建立WspR本身的结构特征，这对于该二鸟苷酸环化酶的亚细胞定位和体内功能是重要的（目的3）。在过去的工作中，已经表明具有可忽略的细胞内c-di-GMP的铜绿假单胞菌细胞不能启动生物膜形成。本文提出的阐明c-di-GMP介导的对细胞生理学的影响的机制的工作对于开发基于c-di-GMP的治疗剂以预防生物膜感染可能是重要的。公共卫生相关性：被称为生物膜的多细胞细菌群落是导致人类慢性感染的原因，具有潜在的易感性，如囊性纤维化。生物膜感染很难用抗生素治疗，并且往往会逃脱人体免疫系统。这项研究将探索一种名为cyclic-di-GMP的小分子如何成为预防或治疗生物膜感染的靶点。

DESCRIPTION (provided by applicant): This project is to determine mechanisms by which the secondary intracellular metabolite, cyclic diguanylate (c-di-GMP), stimulates the formation of biofilms in the opportunistic pathogen Pseudomonas aeruginosa. Biofilms, defined as surface-associated multicellular communities encased in a self-produced extrapolysaccharide (EPS) matrix, are responsible for chronic P. aerugionsa infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are problematic because they are resistant to antibiotic treatment and tend to escape immune surveillance. P. aeruginosa cells with high c-di-GMP form thick biofilms and distinctive wrinkled colonies and have decreased motility. The major biofilm-related effect of c-di-GMP in P. aeruginosa is to stimulate Psl and Pel EPS production. This is partly due to effects of c-di-GMP on pel and psl gene expression. C-di-GMP also stimulates the activities of the EPS biosynthetic enzymes. This effect is likely due to the compartmentalized production of c-di-GMP at specific subcellular sites by specific diguanylate cyclases. Although the general parameters of c-di-GMP activity have been established, the mechanisms of c-di-GMP action are just beginning to be explored. Proposed experiments will test the model that the transcription factor FleQ responds to c-di-GMP in concert with FleN to regulate transcription. An in vitro system with purified proteins and pel promoter DNA will be the starting point for experiments to explore mechanism (aim 1). Other experiments will characterize the catalytic properties of WspR, the most active diguanylate cyclase from P. aeruginosa. WspR is a hybrid response regulator-diguanylate cyclase that synthesizes c-di-GMP when activated by phosphorylation. wspR mutants are defective in EPS synthesis. Constitutively active mutant forms of WspR will be particularly targeted for characterization (aim 2). Fluorescent protein-tagged WspR forms dynamic clusters in the cytoplasm of cells when it is phosphorylated and therefore active. This indicates that WspR-P produces c-di-GMP at discreet subcellular locations and implies that cells have specific WspR-associated targets of c-di-GMP action. The subcellular locations of known c-di-GMP receptor proteins and their possible interactions with WspR will be assessed. Experiments to identify cytoskeleton proteins that may guide the distribution of WspR-P will also carried out. In addition, fluorescence microscopy will be used to establish structural features of WspR itself that are important for the subcellular localization and in vivo function of this diguanylate cyclase (aim 3). In past work it has been shown that P. aeruginosa cells with negligible intracellular c-di-GMP are unable to initiate biofilm formation. The work proposed here to elucidate mechanisms of c-di-GMP-mediated effects on cellular physiology could be important for the development of c-di-GMP based therapeutics to prevent biofilm infections. PUBLIC HEALTH RELEVANCE: Multicellular communities of bacteria called biofilms are responsible for chronic infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are difficult to treat with antibiotics and tend to escape the human immune system. This research will explore how a small molecule called cyclic-di-GMP might be a target for preventing or treating biofilm infections.