Proteogenome of Anaerobic P. aeruginosa in CF Mucus

CF 粘液中厌氧铜绿假单胞菌的蛋白质组

• 批准号: 6609992
• 负责人: DANIEL J. HASSETT
• 金额: $ 33.35万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6609992
• 关键词: Pseudomonas aeruginosa; anaerobiosis; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; biofilm; cell line; confocal scanning microscopy; cystic fibrosis; functional /structural genomics; gene mutation; genetic library; genetic screening; genetic transcription; green fluorescent proteins; liquid chromatography mass spectrometry; matrix assisted laser desorption ionization; microarray technology; microorganism culture; mucus; nitrification; periplasm; polymerase chain reaction; proteomics; transposon /insertion element

DESCRIPTION (provided by applicant): CF patients succumb to pulmonary insufficiency, resulting from complications due to intractable P. aeruginosa biofilms. Because there is no animal model for CF airway disease, in vitro biofilm studies have been performed using a variety of substrata (plastic, glass, stones, etc.) for bacterial attachment and biofilm development. However, unlike abiotic surfaces or even cells, CF airway bacteria are not associated with the epithelium. Instead, they are enmeshed in hypoxic or anaerobic mucopurulent masses in thick mucus lining the airway lumen. Therefore, we hypothesize that P. aeruginosa is undergoing anaerobic respiration (denitrification) during the course of CF airway disease and the process of denitrification is likely critical for optimal growth and survival of the organism in CF airway mucus. Several lines of evidence suggest that these hypotheses are correct. First, antibodies are raised by CF patients against proteins involved in anaerobic metabolism of P. aeruginosa. Second, P. aeruginosa alginate production, that is directly associated with CF morbidity and mortality, and is typically unstable in vitro. Yet it is stabilized during anaerobic growth in the presence of the alternative electron acceptor, nitrate, that is present in ample quantities in CF airway mucus for anaerobic growth of P. aeruginosa. With the completed P. aeruginosa genome and the development of genomics/proteomics, a timely opportunity exists to monitor whole-genome transcription and protein expression patterns under defined conditions that are relevant to the pathogenesis of CF airway disease. The research agenda for this proposal will include a set of experiments using 1- and 2-D gel/MALDI mass spectrometry/proteomics, capillary LCMS mass spectrometry (whole-bacterium proteome analysis), and microarray transcriptional profiling that are organized to fulfill a single primary goal - to identify P. aeruginosa genes and proteins necessary for optimal survival of biofilm bacteria undergoing anaerobic metabolism in mucus derived from CF primary cell lines. The genes/proteins identified in these studies are predicted to mimic those expressed in various stages of CF airway infection. Our LONG TERM GOAL is to identify cellular targets that inhibit or significantly compromise the denitrification process that is occurring by P. aeruginosa in the CF airways.

描述（由申请人提供）：CF患者死于肺功能不全，由难治性铜绿假单胞菌生物膜引起的并发症引起。因为没有CF气道疾病的动物模型，所以已经使用各种基质（塑料、玻璃、石头等）进行了体外生物膜研究。用于细菌附着和生物膜形成。然而，与非生物表面或甚至细胞不同，CF气道细菌与上皮无关。相反，它们被嵌在气道腔厚粘液中的缺氧或厌氧粘液脓性肿块中。因此，我们假设铜绿假单胞菌在CF气道疾病的过程中正在经历厌氧呼吸（脱氮），并且脱氮过程可能对于CF气道粘液中生物体的最佳生长和存活至关重要。几条证据表明这些假设是正确的。首先，CF患者针对参与铜绿假单胞菌厌氧代谢的蛋白质产生抗体。第二，铜绿假单胞菌藻酸盐产生，其与CF发病率和死亡率直接相关，并且通常在体外不稳定。然而，它在厌氧生长期间在替代电子受体硝酸盐的存在下稳定，硝酸盐以充足的量存在于CF气道粘液中用于铜绿假单胞菌的厌氧生长。随着铜绿假单胞菌基因组的完整和基因组学/蛋白质组学的发展，在与CF气道疾病的发病机制相关的限定条件下及时存在监测全基因组转录和蛋白质表达模式的机会。该提案的研究议程将包括一系列使用1-和2-D凝胶/MALDI质谱/蛋白质组学、毛细管LCMS质谱的实验（全细菌蛋白质组分析），和微阵列转录谱分析，它们被组织起来以实现一个单一的主要目标-鉴定在来源于CF的粘液中经历厌氧代谢的生物膜细菌的最佳存活所必需的铜绿假单胞菌基因和蛋白质原代细胞系预测在这些研究中鉴定的基因/蛋白质模拟在CF气道感染的各个阶段中表达的那些。我们的长期目标是确定抑制或显著损害CF气道中铜绿假单胞菌发生的脱氮过程的细胞靶点。