Geobiological approaches to understanding pulmonary infections in situ

描述（由申请人提供）：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并改变它的无知的限制。不幸的是，进行原位研究的方法将提供这样的见解在很大程度上缺乏生物医学，迫切需要开发新的技术方法。传染病研究中现有的主导范式是使用（必然不完美的）模型生物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，生态系统远不止是各部分的总和，物种之间的相互作用（竞争和合作）可以显著影响个体的行为。正因为如此，传统方法可能只能揭示更大图景的一部分。 随着人们对人类微生物组在决定人类健康和疾病方面的重要性的认识不断提高，人们对微生物生态学的认识也在不断提高，但这一领域的研究仍然几乎完全集中在调查身体各部位内存在的微生物群落（例如NIH资助的人类微生物组项目）。在这里，我们提出了开发和应用一些新的方法来回答以下关于囊性纤维化（CF）患者肺部的机会性病原体的问题：i）谁在那里，以及如何在肺内感染？ 它们在空间上相互关联并与宿主关联（二）作为一种功能， 空间和时间的结合和iii）它们利用什么代谢途径？在这样做的过程中，我们将借鉴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。我们将采用最先进的微电极来表征微米尺度的宿主肺的化学环境，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2 H从水中掺入脂质将被开发为原位生长速率的新代理，使用nanoSIMS对15 N掺入蛋白质的空间作图将用于辨别亚微米模式的代谢活性。使用16 S rRNA定向探针的荧光原位杂交（FISH）将使得能够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，我们将开发新的荧光原位杂交探针靶向特定的mRNA转录使用一种新的杂交链反应技术来映射代谢基因的表达。这些方法将使用铜绿假单胞菌的渗透和生物膜培养物进行改进和验证，随后与洛杉矶儿童医院的临床医生合作应用于CF患者样本。南加州大学成人CF诊所咳出的痰液将用于深入了解肺部微生物组的时间演变，而吸痰的肺将用于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传染病的研究。

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.