Bacterial mucin degradation in cystic fibrosis airway disease.

项目摘要/摘要 尽管对囊性纤维化(CF)细菌病原体进行了广泛的研究，但抗生素的疗效 在体外取得的成果很少能转化为成功的临床结果。这种限制在一定程度上是由 我们对细菌适应的宿主环境和代谢策略缺乏了解 在活体内维持群落的生长。更深入地了解什么是CF病原体的研究 在囊性纤维化气管内做是必要的，以提供更有效的治疗策略。 这项建议阐述了CF病原体如何在肺内获得营养。尽管 细菌细胞以高密度存在，维持它们生长的营养来源(S)不是 明显的；主要的碳库，粘蛋白，对典型的 病原菌铜绿假单胞菌。考虑到它们在肺中的丰富和它们的降解能力 唾液粘蛋白，我们认为，通常与口腔相关的厌氧菌，促进碳水化合物 被假单胞菌感染，进而引发疾病。更具体地说，共生细菌(例如： 普雷沃特氏菌、韦洛氏菌)能够依靠呼吸道粘蛋白分泌物茁壮成长，释放混合发酵 副产品。我们假设如果允许堆积(由于粘液清除受损)， 发酵产生的代谢物可以支持其他生物的生长。我们最近展示了 唾液来源的细菌群落在提供粘蛋白时刺激铜绿假单胞菌的生长 唯一的碳源。这表明，粘蛋白降解厌氧菌可能支持病原体在 感染。基于这些数据，该项目将使用多学科方法来(1)确定 厌氧细菌及其产生的支持铜绿假单胞菌的粘蛋白代谢物 生长，(2)使用稳定的同位素标记方法来跟踪粘蛋白衍生的碳在整个过程中的流动 Cf痰，(3)使用粘蛋白过度生产的细胞系评估假单胞菌与上皮细胞的相互作用 粘蛋白降解厌氧菌的存在，以及(4)使用小鼠慢性肺部感染模型 口腔来源的假单胞菌混合感染对体内生长和致病性的影响 厌氧菌。 在这项研究完成时，我们将确定口腔相关细菌的致病作用。 并提供了CF肺内碳流动的详细特征。总而言之， 这些研究将对我们理解纤维结合蛋白的功能作用产生有意义的影响 微生物区系和治疗策略的潜在发展。而CF微生物组将服务于 作为研究粘蛋白衍生的交叉喂养相互作用的起点，我们的方法同样是 适用于预防其他呼吸系统疾病-慢性阻塞性肺病、慢性鼻窦炎和呼吸机- 相关性肺炎--粘性粘液环境中存在复杂的细菌感染。

PROJECT SUMMARY / ABSTRACT Despite extensive studies of cystic fibrosis (CF) bacterial pathogens, the efficacy of antibiotics achieved in vitro seldom translates to successful clinical outcomes. This limitation is partially driven by our lack of understanding of the host environments to which bacteria adapt, and the metabolic strategies that sustain community growth in vivo. Studies that generate greater insight into what CF pathogens are doing within the cystic fibrosis airways are necessary to inform more effective therapeutic strategies. This proposal addresses how CF pathogens obtain nutrients within the lung. Despite the presence of bacterial cells at high densities, the nutrient source(s) that sustains their growth is not apparent; the predominant carbon reservoir, mucins, are recalcitrant to degradation by the canonical pathogen Pseudomonas aeruginosa. Given their abundance in the lung and their ability to degrade salivary mucins, we propose that anaerobes typically associated with the oral cavity, facilitate carbon acquisition by Pseudomonas, which in turn drives disease. More specifically, commensal bacteria (e.g. Prevotella, Veillonella) are able to thrive off respiratory mucin secretions, releasing mixed fermentation byproducts. We hypothesize that if allowed to accumulate (due to impaired mucus clearance), fermentation-derived metabolites can support the growth of other organisms. We have recently shown that saliva-derived bacterial communities stimulate the growth of P. aeruginosa when provided mucin as the sole carbon source. This suggests that mucin-degrading anaerobes may support pathogens during infection. Based on these data, this project will use a multi-disciplinary approach to (1) identify the anaerobic bacteria and the mucin-derived metabolites they generate that can support P. aeruginosa growth, (2) Use a stable isotope labeling approach to track the flow of mucin-derived carbon throughout CF sputum, (3) Use a mucin-overproducing cell line to assess Pseudomonas-epithelium interactions in the presence of mucin-degrading anaerobes, and (4) use a murine chronic lung infection model to assess the growth and pathogenicity of Pseudomonas in vivo when co-infected with oral-derived anaerobes. At the completion of this study, we will have defined a pathogenic role for oral-associated bacteria in the lower airways and provide a detailed characterization of carbon flow within the CF lung. Altogether, these studies will have a meaningful impact on our understanding of the functional role of the CF microbiota and the potential development of therapeutic strategies. While the CF microbiome will serve as a starting point to investigate mucin-derived cross-feeding interactions, our approach is equally applicable to the prevention of other respiratory diseases – COPD, chronic sinusitis, and ventilator- associated pneumonias – where a viscous mucus environment harbors complex bacterial infections.