The role of anaerobic microbiota in cystic fibrosis airway disease trajectory

项目摘要/摘要 尽管最近关于厌氧菌的流行率和丰度的研究激增 囊性纤维化(CF)患者呼吸道中的微生物群，如果有的话，它们对 疾病的病理生理学仍存在争议，且知之甚少。这种知识鸿沟是由 部分，由于流行病学和体外实验数据相互矛盾，缺乏合适的模型系统进行研究 它们与宿主的相互作用，以及缺乏与宿主相关的厌氧实验室专业知识 微生物学。更深入地了解厌氧菌在慢性支气管炎肺部感染中的作用， 特别是在早期阶段，对于确定是否、何时以及如何针对这组生物是必要的 从治疗上讲。 我们的团队已经证明：(I)厌氧菌是儿科CF呼吸道的早期定居者，在 获得典型病原体，(Ii)厌氧菌群落引发促炎反应 当与支气管上皮细胞共培养时，以及(Iii)厌氧菌介导的粘蛋白降解和交叉 喂食可以影响典型的CF病原体的生长、毒力和对抗生素的敏感性。建房 根据这些数据，该项目建议使用多学科方法来调查个人如何 厌氧菌与彼此、呼吸道上皮和传统病原体(例如，铜绿假单胞菌， 金黄色葡萄球菌)在早期慢性支气管炎呼吸道疾病的背景下。首先，使用一个实验性的微生物组模型 系统由10个碳纤维厌氧菌组成，我们将确定碳纤维细菌群落是如何组装的 以及它们的组装动态是如何由宿主和微生物过程塑造的。第二，使用 创新的双重缺氧系统，可实现厌氧细菌和呼吸道上皮细胞的共培养， 我们将使用单细胞RNA测序来确定厌氧菌如何与呼吸道上皮相互作用， 以及宿主反应如何随上皮细胞类型的不同而变化。最后，鉴于铜绿假单胞菌、金黄色葡萄球菌和 其他需氧病原菌与厌氧微生物群共同定植，并作为患者迅速适应呼吸道 年龄，我们将使用体外实验进化方法来测试早期CF微生物群是否 足以驱动典型病原体适应慢性阻塞性肺疾病呼吸道的营养环境。 在完成这些目标时，我们将定义单个厌氧菌如何与 另一种是寄主和传统病原菌。通过这样做，这些研究将产生有意义的影响 关于我们对呼吸道微生物区系在慢性呼吸道疾病中作用的机械理解，并可能 推动未来针对特定厌氧菌的研究，甚至促进它们的生长，作为一种手段 改善临床结果。最后，我们使用CF作为我们的模型系统，但这项工作具有广泛的适用性 其他急性和呼吸道感染(如慢性阻塞性肺病、鼻窦炎、呼吸机相关性肺炎) 厌氧菌已经被牵连，但它们的机制贡献仍然不确定。

PROJECT SUMMARY / ABSTRACT Despite the recent surge in studies reporting on the prevalence and abundance of anaerobic microbiota in the airways of individuals with cystic fibrosis (CF), their mechanistic contributions, if any, to disease pathophysiology remain controversial and poorly understood. This knowledge gap is driven, in part, by conflicting epidemiological and in vitro experimental data, a lack of suitable model systems to study their interactions with the host, and the dearth of laboratory expertise in host-associated anaerobic microbiology. Studies that generate greater insight into the role of anaerobes in CF lung infection, particularly at early stages, are necessary to determine if, when, and how to target this group of organisms therapeutically. Our team has shown that (i) anaerobes are early colonizers of the pediatric CF airways prior to the acquisition of canonical pathogens, (ii) anaerobe bacterial communities elicit pro-inflammatory response when co-cultured with bronchial epithelial cells, and (iii) anaerobe-mediated mucin degradation and cross- feeding can shape the growth, virulence, and antibiotic susceptibility of canonical CF pathogens. Building on these data, this project proposes to use a multi-disciplinary approach to investigate how individual anaerobes interact with one another, the airway epithelium, and traditional pathogens (e.g., P. aeruginosa, S. aureus) in the context of early CF airway disease. First, using an experimental microbiome model system composed of 10 CF anaerobic species, we will determine how CF bacterial communities assemble and how their assembly dynamics are shaped by host and microbial processes. Second, using an innovative dual oxic-anoxic system that enables co-culture of anaerobic bacteria and airway epithelial cells, we will use single-cell RNA sequencing to determine how anaerobes interact with the airway epithelium, and how the host response varies by epithelial cell type. Finally, given that P. aeruginosa, S. aureus, and other aerobic pathogens co-colonize with anaerobic microbiota and rapidly adapt to the airways as patients age, we will use an in vitro experimental evolution approach to test whether early CF microbiota are sufficient to drive the adaptation of canonical pathogens to the nutritional milieu of the CF airways. At the completion of these aims, we will have defined how individual anaerobes interact with one another, the host, and traditional CF pathogens. In doing so, these studies will have a meaningful impact on our mechanistic understanding of the role of airway microbiota in chronic airway disease and may motivate future studies targeting specific anaerobic bacteria, or even promoting their growth, as a means of improving clinical outcomes. Finally, we use CF as our model system, but this work is broadly applicable to other acute and airway infections (e.g., COPD, sinusitis, ventilator associated pneumonias) in which anaerobes have been implicated but their mechanistic contributions remain undefined.