Cyclic-di-GMP signaling in Pseudomonas aeruginosa

铜绿假单胞菌中的环二 GMP 信号传导

• 批准号: 8000041
• 负责人: Caroline Stone Harwood
• 金额: $ 7.91万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000041
• 关键词: Agonist; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Cell physiology; Cells; Characteristics; Chemotaxis; Chronic; Communities; Complex; Cystic Fibrosis; Cytoplasm; Cytoskeleton; DNA; DNA-Protein Interaction; Data; Development; Enzymes; Fluorescence Microscopy; Gene Expression; Genetic Transcription; Growth; Human; Hybrids; Immune system; Immunologic Surveillance; In Vitro; Infection; Kinetics; Location; Mediating; Microbial Biofilms; Modeling; Movement; Outcome; Phosphorylation; Predisposition; Production; Property; Proteins; Pseudomonas aeruginosa; Repression; Research; Set protein; Signal Transduction; Site; Solid; Stimulus; Surface; System; Testing; Therapeutic; Thick; Transcriptional Regulation; Work; base; bis(3' ,5' )-cyclic diguanylic acid; cell motility; diguanylate cyclase; in vitro activity; in vivo; inorganic phosphate; mutant; pathogen; prevent; promoter; protein-histidine kinase; public health relevance; receptor; reconstitution; research study; response; small molecule; transcription factor

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.

描述（由申请人提供）：该项目旨在确定次级细胞内代谢物环二鸟苷酸（c-di-GMP）刺激机会性病原体铜绿假单胞菌中生物膜形成的机制。生物膜被定义为包裹在自身产生的外多糖 (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倾向的人类慢性铜绿假单胞菌感染的原因。生物膜感染是一个问题，因为它们对抗生素治疗有抵抗力，并且往往会逃避免疫监视。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皱纹集落，并且运动性降低。 c-di-GMP 在铜绿假单胞菌中的主要生物膜相关作用是刺激 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。尽管c-di-GMP活性的一般参数已经建立，但c-di-GMP作用机制才刚刚开始探索。拟议的实验将测试转录因子 FleQ 响应 c-di-GMP 与 FleN 协同调节转录的模型。具有纯化蛋白质和 pel 启动子 DNA 的体外系统将成为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 是来自铜绿假单胞菌的最活跃的二鸟苷酸环化酶。 WspR 是一种混合反应调节剂-二鸟苷酸环化酶，当被磷酸化激活时可合成 c-di-GMP。 wspR 突变体在 EPS 合成中存在缺陷。 WspR 的组成型活性突变体形式将成为表征的特别目标（目标 2）。当荧光蛋白标记的 WspR 被磷酸化并因此活跃时，它会在细胞的细胞质中形成动态簇。这表明 WspR-P 在离散的亚细胞位置产生 c-di-GMP，并暗示细胞具有特定的 WspR 相关 c-di-GMP 作用靶点。将评估已知 c-di-GMP 受体蛋白的亚细胞位置及其与 WspR 可能的相互作用。还将开展鉴定可指导 WspR-P 分布的细胞骨架蛋白的实验。此外，荧光显微镜将用于建立 WspR 本身的结构特征，这对于这种二鸟苷酸环化酶的亚细胞定位和体内功能非常重要（目标 3）。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无法启动生物膜形成。这里提出的阐明 c-di-GMP 介导的细胞生理学影响机制的工作对于开发基于 c-di-GMP 的预防生物膜感染的疗法可能很重要。公共卫生相关性：被称为生物膜的多细胞细菌群落是导致具有囊性纤维化等潜在倾向的人类慢性感染的原因。生物膜感染很难用抗生素治疗，并且往往会逃避人体免疫系统。这项研究将探索一种称为环二 GMP 的小分子如何成为预防或治疗生物膜感染的靶标。