Proteogenome of Anaerobic P. aeruginosa in CF Mucus

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。由于没有CF气道疾病的动物模型，因此使用各种基质（塑料，玻璃，石头等）进行了体外生物膜研究，以进行细菌附着和生物膜发育。然而，与非生物表面甚至细胞不同，CF气道细菌与上皮无关。相反，它们被困在气道腔内厚粘液中的缺氧或厌氧粘液化脓性肿块中。因此，我们假设P. aeruginosa在CF气道疾病过程中正在进行厌氧呼吸（反硝化），而反硝化过程可能对CF气道粘液中生物的最佳生长和生存至关重要。一些证据表明这些假设是正确的。首先，CF患者会产生针对铜绿假单胞菌厌氧代谢相关蛋白的抗体。其次，P. aeruginosa海藻酸盐的产生与CF发病率和死亡率直接相关，并且在体外通常不稳定。然而，在厌氧生长过程中，在替代电子受体硝酸盐的存在下，它是稳定的，硝酸盐在铜绿假单胞菌厌氧生长的CF气道粘液中大量存在。随着铜绿假单胞菌基因组的完成和基因组学/蛋白质组学的发展，在特定条件下监测与CF气道疾病发病机制相关的全基因组转录和蛋白质表达模式是一个及时的机会。该提案的研究议程将包括一系列使用1- d和2-D凝胶/MALDI质谱/蛋白质组学，毛细管LCMS质谱（全细菌蛋白质组分析）和微阵列转录谱的实验，这些实验旨在实现一个主要目标-鉴定铜绿假单胞菌基因和蛋白质，这些基因和蛋白质对于在CF原代细胞系的粘液中进行厌氧代谢的生物膜细菌的最佳存活是必要的。在这些研究中发现的基因/蛋白被预测与CF气道感染不同阶段的基因/蛋白表达相似。我们的长期目标是确定抑制或显著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点。

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.