Cyclic-di-GMP signaling in Pseudomonas aeruginosa

描述(由申请人提供)：本项目旨在确定次生胞内代谢产物环二鸟苷(c-di-GMP)刺激条件致病菌铜绿假单胞菌生物被膜形成的机制。生物膜是包裹在自身产生的胞外多糖(EPS)基质中的表面相关的多细胞群落，是人类慢性铜绿假单胞菌感染的罪魁祸首，具有潜在的易感性，如囊性纤维化。生物被膜感染是有问题的，因为它们对抗生素治疗具有抵抗力，并且往往逃脱免疫监视。高c-di-GMP的铜绿假单胞菌细胞形成厚厚的生物膜和独特的皱纹菌落，并降低了活力。C-di-GMP对铜绿假单胞菌生物被膜的主要作用是刺激PSL和Pel-EPS的产生。这部分是由于c-di-GMP对Pel和PSL基因表达的影响。C-di-GMP还能刺激EPS生物合成酶的活性。这种影响可能是由于特定的二鸟苷酸环化酶在特定的亚细胞位置产生c-di-GMP所致。虽然c-di-GMP活性的一般参数已经建立，但c-di-GMP的作用机制才刚刚开始探索。拟议的实验将测试转录因子FleQ与FleN协同反应c-di-GMP以调节转录的模型。由纯化的蛋白质和PEL启动子DNA组成的体外系统将是探索机制的实验的起点(目标1)。其他实验将表征WSPR的催化特性，WSPR是铜绿假单胞菌最活跃的二鸟苷环化酶。WSPR是一种混合型反应调节因子--二鸟苷环化酶，被磷酸化激活后合成c-di-GMP。WSPR突变体在EPS合成方面存在缺陷。WSPR的构成活性突变形式将特别针对特征(目标2)。当荧光蛋白标记的WSPR被磷酸化时，它在细胞质中形成动态簇，因此是活性的。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作用的特定WSPR相关靶点。已知的c-di-GMP受体蛋白的亚细胞位置以及它们可能与WSPR的相互作用将被评估。还将进行实验，以确定可能指导WSPR-P分布的细胞骨架蛋白。此外，荧光显微镜将被用来建立WSPR本身的结构特征，这些结构特征对于这种二鸟苷酸环化酶(AIM 3)的亚细胞定位和体内功能是重要的。在过去的工作中已经表明，细胞内c-di-GMP可以忽略不计的铜绿假单胞菌细胞不能启动生物膜的形成。本研究旨在阐明c-di-GMP对细胞生理的影响机制，这对于开发基于c-di-GMP的预防生物被膜感染的药物具有重要意义。与公共卫生相关：称为生物膜的多细胞细菌群落是人类慢性感染的罪魁祸首，具有囊性纤维化等潜在倾向。生物被膜感染很难用抗生素治疗，而且往往会逃脱人类免疫系统。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被膜感染的靶点。

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.