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患者因棘手的铜绿假单胞菌生物膜而造成的并发症导致肺部功能不全。由于没有用于CF气道疾病的动物模型，因此已经使用各种基质（塑料，玻璃，石材等）进行了体外生物膜研究，以进行细菌附着和生物膜发育。但是，与非生物表面甚至细胞不同，CF气道细菌与上皮无关。取而代之的是，它们以低氧或厌氧性粘液质量的浓度诱捕，在气道管腔内厚的粘液中。因此，我们假设铜绿假单胞菌在CF气道疾病过程中正在接受厌氧呼吸（反硝化），而硝化过程对于CF气道粘液中有机体的最佳生长和生存至关重要。几条证据表明这些假设是正确的。首先，CF患者对参与铜绿假单胞菌厌氧代谢的蛋白质提出抗体。其次，与CF发病率和死亡率直接相关的铜绿假单胞菌藻酸盐产生，通常在体外不稳定。然而，在存在替代电子受体，硝酸盐的存在下，它在厌氧生长过程中稳定，在CF气道粘液中以足够数量的含量存在，用于铜绿假单胞菌的厌氧生长。随着完整的铜绿假单胞菌基因组和基因组/蛋白质组学的发展，存在及时的机会来监测与CF气道疾病发病机理有关的定义条件下的全基因组转录和蛋白质表达模式。该提案的研究议程将包括一组实验，使用1-和2-D凝胶/MALDI质谱/蛋白质组学，毛细管LCMS质谱法（全-细菌蛋白质组分析）和微阵列转录分析，以实现单个单一的转录分析主要目标 - 鉴定铜绿假单胞菌基因和蛋白质，是在CF主要细胞系衍生的粘液中经历厌氧代谢的生物膜细菌最佳存活所必需的。这些研究中鉴定出的基因/蛋白质预测将模仿在CF气道感染的各个阶段表达的基因。我们的长期目标是确定抑制或显着损害铜绿假单胞菌在CF气道中发生的反硝化过程的细胞靶标。