Cyclic-di-GMP signaling in Pseudomonas aeruginosa

描述（由申请人提供）：本项目旨在确定细胞内次级代谢物环二胍酸酯（c-di-GMP）刺激机会致病菌铜绿假单胞菌生物膜形成的机制。生物膜被定义为包裹在自产外多糖（EPS）基质中的表面相关多细胞群落，是导致具有囊性纤维化等潜在易感性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具有耐药性，并且往往逃避免疫监视。具有高c-二gmp的铜绿假单胞菌细胞形成厚的生物膜和独特的皱褶菌落，并降低运动性。c-di-GMP在铜绿假单胞菌中与生物膜相关的主要作用是刺激Psl和Pel EPS的产生。这部分是由于c-二- gmp对pel和psl基因表达的影响。c -二gmp还能刺激EPS生物合成酶的活性。这种效应可能是由于特定的二胍酸环化酶在特定的亚细胞位点区隔化生产c-二gmp。虽然c-di-GMP活性的一般参数已经确定，但对c-di-GMP作用机制的探索才刚刚开始。拟议的实验将测试转录因子FleQ与FleN一起响应c-di-GMP以调节转录的模型。一个含有纯化蛋白和pel启动子DNA的体外系统将是探索机制的实验起点（目的1）。其他实验将表征WspR的催化性能，WspR是铜绿假单胞菌中最活跃的二胍酸环化酶。WspR是一种混合反应调节因子-二胍酸环化酶，当被磷酸化激活时合成c-二gmp。wspR突变体在EPS合成上有缺陷。组成型活性突变型的WspR将特别针对表征（目的2）。当荧光蛋白标记的WspR被磷酸化并因此具有活性时，它在细胞的细胞质中形成动态簇。这表明wsp - p在离散的亚细胞位置产生c-di-GMP，并暗示细胞具有特定的wsp - p相关的c-di-GMP作用靶点。将评估已知c-二gmp受体蛋白的亚细胞位置及其与WspR可能的相互作用。鉴定可能指导wspp分布的细胞骨架蛋白的实验也将进行。此外，荧光显微镜将用于确定WspR本身的结构特征，这些特征对这种二胍酸环化酶的亚细胞定位和体内功能很重要（目的3）。在过去的研究中已经表明，胞内c-二gmp可忽略不计的铜绿假单胞菌细胞不能启动生物膜的形成。本文提出的阐明c-di-GMP介导的细胞生理作用机制的工作，可能对开发基于c-di-GMP的治疗方法来预防生物膜感染具有重要意义。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潜在易感性，如囊性纤维化。生物膜感染很难用抗生素治疗，而且往往会逃离人体免疫系统。这项研究将探索一种名为环二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.