Testing the hypothesis that microbial energetic hijacking of the CF immune response selects for specific pathogens during lung function decline

项目总结 患有囊性纤维化(CF)的患者与毁灭性的慢性呼吸道微生物感染作斗争。 尽管慢性萎缩性胃炎患者通常会被其他无害的细菌和真菌定植--其中许多来自 从土壤环境中-受损的CF免疫系统无法清除这些机会主义者，并且 随之而来的宿主和微生物之间的斗争最终导致肺部系统衰竭。传统型 抗生素不是很有效。对于为什么会出现这种情况，一个被广泛接受的观点是，栖息的微生物区系 生长缓慢，它们的膜没有足够的电荷来吸收常用的抗生素 作为妥布霉素。因此，如果我们寻求治疗肺部感染的新方法，我们必须更好地了解如何 微生物在这个栖息地繁衍生息。不同的证据表明，氧气在慢性阻塞性肺疾病的呼吸道中是有限的 与机会性病原体相关的微观尺度。虽然碳源充满在充满粘液的 航空公司，在没有氧气的情况下，主导这个栖息地的细菌和真菌必须利用 有氧呼吸的替代能源产生策略。他们怎么做到这一点的？能量流是驱动力 任何微生物群落的组织者，包括在CF航空中发现的微生物群落。公认的是， CF患者免疫系统产生的一氧化氮(NO)低于健康人 个人。事实上，上皮细胞在没有产生的情况下受到严重损害，但中性粒细胞充斥着 Cf呼吸道仍然可以产生一氧化氮；这种一氧化氮不足以杀死微生物，但有可能转化为 变成了那些有能力进行反硝化的人的能源。因此，我们试图检验这一假设 微生物通过选择特定的反硝化途径劫持CFs免疫反应 肺功能下降期间的病原体。为了验证这一假设，我们计划利用强大的同位素工具 来自允许复杂环境中的代谢物来源的地球科学，如CF Airways，将逐个原子进行测定，提供非侵入性的法医分子指纹。特别是， 我们将重点解释N2O，它是CF痰和呼吸中反硝化途径的可测量产物 汽油。除了使用细菌生理学、遗传学和宿主微生物的原位成像的方法之外 相互作用，我们将使用这些同位素工具来深入了解在CF中NO是如何还原为N2O的 航空公司可能会偏爱特定微生物群落成员的健康。为此，我们提出了三个具体的建议 目标：目标1将确定导致普通CF细菌产生N2O的条件和途径 真菌，目标2将利用先进的同位素分析来剖析这些微生物产生的N2O，以 取证地推断其来源，Aim 3将把这些见解应用于一项针对成年CF患者的试点研究，以确定 随着肺功能的下降，特定类型的反硝化细菌是否受到青睐。实现这些目标将 提供必要的关键知识，以指导早期治疗CF感染的翻译方法 以及建立可更广泛地应用于其他生物失调研究的工具。

PROJECT SUMMARY Individuals living with cystic fibrosis (CF) combat devastating, chronic microbial infections of the airways. Though CF patients are usually colonized by otherwise innocent bacteria and fungi—many of which derive from soil environments—the compromised CF immune system is unable to clear these opportunists, and the ensuing struggle between host and microbes eventually leads to failure of the pulmonary system. Conventional antibiotics are not very effective. One widely held view for why this is the case is that the resident microbiota are growing slowly and their membranes are insufficiently charged to take up commonly-used antibiotics such as tobramycin. Accordingly, if we seek new ways to treat CF lung infections, we must better understand how microbes thrive in this habitat. Different lines of evidence indicate that oxygen is limiting in the CF airways at the microscale relevant to opportunistic pathogens. Though carbon sources are replete in the mucus-filled airways, in the absence of oxygen, the bacteria and fungi that come to dominant this habitat must employ alternative energy generation strategies to aerobic respiration. How do they do this? Energy flow is the driving organizer of any microbial community, including those found in the CF airways. It is well established that the nitric oxide (NO) generated by the immune system in the CF patients is lower than that made by healthy individuals. Indeed, epithelial cells are severely compromised in NO production, yet neutrophils that flood the CF airways can still generate NO; this NO is insufficient to kill the microbes, but has the potential to transform into an energy source for those capable of denitrification. Accordingly, we seek to test the hypothesis that microbial energetic hijacking of the CF immune response via denitrification pathways selects for specific pathogens during lung function decline. To test this hypothesis, we plan to leverage powerful isotopic tools from the Earth sciences that permit the sources of metabolites in complex environments, such as the CF airways, to be determined atom by atom, providing a non-invasive, forensic molecular fingerprint. In particular, we will focus on interpreting N2O, a measurable product of the denitrification pathway in CF sputum and breath gas. In addition to approaches employing bacterial physiology, genetics, and in situ imaging of host-microbe interactions, we will use these isotopic tools to gain insight into how the NO that is reduced to N2O in the CF airways may favor the fitness of particular microbial community members. To do this, we propose three specific aims: Aim 1 will identify the conditions and pathways leading to N2O production by common CF bacteria and fungi, Aim 2 will utilize advanced isotopic analyses to dissect the N2O produced by these microbes to forensically infer its source, and Aim 3 will apply these insights to a pilot study of adult CF patients to determine whether particular types of denitrifiers are favored as lung function declines. Attainment of these objectives will provide the critical knowledge needed to guide early translational approaches for treating infections of the CF airways, as well as establish tools that can be applied more broadly to other studies of dysbiosis.