Geobiological approaches to understanding pulmonary infections in situ

描述(申请人提供)：许多传染病的有效治疗受到对感染环境以及病原体如何适应和改变感染环境的无知的限制。不幸的是，生物医学中缺乏进行这种洞察的现场研究方法，迫切需要开发新的技术方法。现有的在传染病研究中占主导地位的范式是使用(必然不完美的)模型生物体的还原实验来研究它们的生理学，从而确定新的药物靶点。然而，生态系统远不止是它们各部分的总和，物种之间的相互作用(包括竞争和合作)可以显著影响个体的行为。正因为如此，传统的方法可能只揭示了更大图景的一部分。 人们日益认识到人类微生物组在决定人类健康和疾病方面的重要性，从而增加了对微生物生态学的认识，但这一领域的研究仍然几乎完全集中于调查人体不同部位存在的微生物群落(例如，由美国国立卫生研究院资助的人类微生物组项目)。在这里，我们建议开发和应用一些新的方法来回答以下关于囊性纤维化(CF)患者肺部条件致病菌的问题：i)谁在那里，如何 它们在空间上相互关联，并与宿主相关联？二)作为一项职能，它们的活跃程度如何 既有空间又有时间？以及iii)他们利用的是什么代谢途径？在这样做时，我们将利用我们在地球生物学领域的经验，使用最初开发的工具来跟踪和了解深海沉积物等偏远栖息地的微生物。我们将使用最先进的微电极在微米长的尺度上表征宿主肺的化学环境，寻找塑造微生物生态系统行为和/或由其产生的化学梯度。将水中的2H掺入脂质中作为原位生长速度的新指标，并将使用纳米SIMS将15N掺入蛋白质的空间定位用于识别亚微米级的代谢活动模式。利用16S rRNA定向探针的荧光原位杂交(FISH)技术可以在单细胞水平上确定生物膜内不同细菌物种之间的空间组织。最后，我们将使用一种新的杂交链式反应技术来定位代谢基因的表达，以开发针对特定mRNA转录本的新的FISH探针。这些方法将利用铜绿假单胞菌的浮游和生物膜培养进行提炼和验证，并随后与洛杉矶儿童医院和南加州大学成人CF诊所的临床医生合作应用于CF患者样本。咳出的痰将被用来洞察肺微生物群的时间演变，而移植的肺将被用来研究其空间代谢组织。虽然CF微生物组将作为我们开发这些新方法的起点，但它们最终有可能改变从结核病到疟疾等各种类型的传染病的研究。 公共卫生相关性：我们对病原体在感染的不同阶段如何在人体宿主中存活的无知，严重限制了有效治疗传染病的设计。传统上，试图洞察这个问题的研究都是在实验室中使用模型系统和条件进行的，这些系统和条件不能完美地模拟人类宿主。在这里，我们建议使用一套新的方法直接在原位测量病原体的化学、结构和代谢活性。

DESCRIPTION (provided by applicant): Effective treatment of many infectious diseases is limited by ignorance of the infected environment and how pathogens adapt to it and alter it. Unfortunately, methods to conduct in situ studies that would provide such insight are largely absent from biomedicine, and there is an urgent need to develop new technical approaches. The existing, dominant paradigm in infectious disease research is to use (necessarily imperfect) reductionist experiments with model organisms to study their physiology and thereby identify new drug targets. Yet ecosystems are much more than the sum of their parts, and interactions (both competitive and cooperative) between species can significantly affect the behavior of individuals. Because of this, traditional approaches likely reveal only part of the bigger picture. Growing awareness of the importance of the human microbiome in determining human health and disease has resulted in an increased appreciation for microbial ecology, yet studies in this area still have focused almost entirely on surveying the microbial communities present within various parts of the body (e.g. the NIH-funded Human Microbiome Project). Here we proposed to develop and apply a number of new approaches to answer the following questions about opportunistic pathogens in the lungs of cystic fibrosis (CF) patients: i) who is there, and how are they spatially associated with each other and with the host? ii) how active are they, as a function of both space and time? and iii) what metabolic pathways are they utilizing? In doing so we will draw on our experience in the field of geobiology, using tools originally developed to track and understand microbes in remote habitats like deep-sea sediments. We will employ state- of-the-art microelectrodes to characterize the chemical environment of the host lung at micrometer length scales, looking for chemical gradients that shape the behavior of the microbial ecosystem and/or are generated by it. Incorporation of 2H from water into lipids will be developed as a novel proxy for in situ growth rates, and spatial mapping of 15N incorporation into proteins using nanoSIMS will be used to discern sub-micrometer patterns of metabolic activity. Fluorescence in situ hybridization (FISH) using 16S rRNA-directed probes will enable the spatial organization between different bacterial species within biofilms to be determined at the level of single cells. Finally, we will develop new FISH probes targeting particular mRNA transcripts using a novel hybridization chain-reaction technique to map metabolic gene expression. These methods will be refined and validated using planktonic and biofilm cultures of Pseudomonas aeruginosa and subsequently applied to CF patient samples in collaboration with clinicians from Children's Hospital L.A. and the USC Adult CF Clinic. Expectorated sputum will be used to gain insight into the temporal evolution of the lung microbiome, whereas explanted lungs will be used to study its spatiometabolic organization. While the CF microbiome will serve as our starting point for the development of these new methods, they ultimately have the potential to transform the study of diverse types of infectious diseases ranging from tuberculosis to malaria. PUBLIC HEALTH RELEVANCE: The design of effective therapeutics to combat infectious diseases is profoundly limited by our ignorance of how pathogens survive in the human host at different stages of infection. Traditionally, studies attempting to gain insight into this problem have been performed in the laboratory using model systems and conditions that imperfectly mimic the human host. Here, we propose to directly measure the chemistry, structure and metabolic activity of pathogens in situ using a novel suite of approaches.