c-diGMP Signal Transduction Proteins are Biofilm Drug Targets

描述（由申请人提供）：项目摘要/摘要：囊性纤维化（CF）是一种遗传性疾病，影响美国30，000多人的肺部主要气道。80%的CF患者的改变的气道受到铜绿假单胞菌的持续细菌定植的困扰。该病原体是从CF肺分离的终末病原体之一，与广泛的肺损伤高度相关。铜绿假单胞菌在CF肺中使用常规抗生素疗法极难根除。这导致下呼吸道的反复细菌感染，这是发病率和死亡率的主要原因。几条证据表明，铜绿假单胞菌在CF肺的定殖涉及生物膜生长模式。最重要的是，生物膜对抗生素和杀生物剂的耐药性比嗜热菌高1000倍。在抗生素药物的发现和开发中尚未考虑生物膜生长模式。在初步工作中，我们已经确定了调节生物膜形成所需的潜在目标。这些新靶点是含有GGDEF（二鸟苷酸环化酶）和EAL（磷酸二酯酶）基序的蛋白质，其参与利用第二信使环状diGMP的信号转导途径。这些蛋白质代表了一类具有生物化学活性的新型药物靶点，这些靶点适合于高通量筛选测定。我们的总体目标是发现和开发抑制铜绿假单胞菌生物膜生长模式的药物。我们实现这一目标的策略是鉴定生物膜形成所必需的含GGDEF/EAL基序的蛋白质，验证它们作为抗生物膜药物发现的靶点，并鉴定抑制它们并破坏铜绿假单胞菌中生物膜形成和/或维持的小分子。在本研究中，我们将实现以下具体目标：（1）鉴定影响铜绿假单胞菌PA 01生物膜形成调控的GGDEF/EAL突变体。(2)验证调节c-diGMP水平所需的GGDEF/EAL蛋白的活性对于特异性调节几种铜绿假单胞菌分离株中的生物膜形成至关重要。(3)鉴定c-diGMP依赖性信号通路的其他组分及其效应物，其参与调节经验证的GGDEF/EAL生物膜靶标的生物膜形成。项目叙述：阻止最流行的CF病原体采用生物膜生长模式和发展内在抗生素耐药性机制的药物的开发将代表CF治疗的重大进展。这将使临床医生能够使用这些生物膜阻断药物和临床使用的抗生素的组合来预防或根除囊性纤维化患者的持续感染。这一发展将代表CF肺病治疗的重要一步，并将产生深远的经济和社会影响;使临床医生能够更好地控制困扰CF患者的生物膜相关感染。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Cystic Fibrosis (CF) is a genetic disorder that affects the major airways of the lungs of more than 30,000 people in the United States. The altered airways of 80% of CF patients are afflicted with a persistent bacterial colonization by Pseudomonas aeruginosa. This pathogen is 1 of the terminal pathogens isolated from the CF lung and is highly associated with extensive pulmonary damage. P. aeruginosa is extremely difficult to eradicate in the CF lung using conventional antibiotic therapies. This results in recurrent bacterial infections of the lower respiratory tract, which are the primary cause of morbidity and mortality. Several lines of evidence indicate that colonization of the CF lung by P. aeruginosa involves the biofilm mode of growth. Most significantly, is that biofilms are up to 1000-fold more resistant to antibiotics and biocides than are planktonic bacteria. The biofilm mode of growth has not been considered in antibiotic drug discovery and development. In preliminary work, we have identified potential targets that are required for regulating biofilm formation. These novel targets are proteins containing GGDEF (diguanylate cyclase) and EAL (phosphodiesterase) motifs, which are involved in signal transduction pathways that utilize the secondary messenger cyclic-diGMP. These proteins represent a class of novel drug targets with biochemical activities that are amenable to high-throughput screening assays. Our overall goal is to discover and develop drugs that inhibit the biofilm mode of growth in P. aeruginosa. Our strategy for reaching this goal is to identify GGDEF/EAL motif-containing proteins essential for biofilm formation, validate them as targets for anti-biofilm drug discovery, and identify small molecules that inhibit them and disrupt biofilm formation and/or maintenance in P. aeruginosa. In this proposal, we will achieve the following specific aims: (1) Identify GGDEF/EAL mutants that affect the regulation of biofilm formation in P. aeruginosa PA01. (2) Verify that the activities of GGDEF/EAL proteins required for modulating c-diGMP levels are essential for specifically regulating biofilm formation in several isolates of P. aeruginosa. (3) Identify additional components of the c-diGMP-dependent signaling pathway(s) and their effectors that are involved in regulating biofilm formation for verified GGDEF/EAL biofilm targets. Project Narrative: The development of drugs that block the most prevalent CF pathogens from adopting the biofilm mode of growth and developing intrinsic antibiotic resistance mechanisms will represent a significant advance in the treatment of CF. This will enable clinicians to prevent or to eradicate persistent infections in Cystic Fibrosis patients using a combination of these biofilm-blocking drugs and an antibiotic that is in clinical use. This development will represent a major step forward in the treatment of CF lung disease, and will have a profound economic and social impact; enabling clinicians to better control the biofilm-related infections that plague CF patients.