Department of Microbiology & Immunology, University of Minnesota, 689 23rd Avenue SE, Minneapolis, MN, 55455, United States.
Academic Health Center, University Flow Cytometry Resource, University of Minnesota, 6th St SE, Minneapolis, MN, 55455, United States.
Nat Commun. 2020 May 8;11(1):2287. doi: 10.1038/s41467-020-16163-2.
Culture-independent studies of cystic fibrosis lung microbiota have provided few mechanistic insights into the polymicrobial basis of disease. Deciphering the specific contributions of individual taxa to CF pathogenesis requires comprehensive understanding of their ecophysiology at the site of infection. We hypothesize that only a subset of CF microbiota are translationally active and that these activities vary between subjects. Here, we apply bioorthogonal non-canonical amino acid tagging (BONCAT) to visualize and quantify bacterial translational activity in expectorated sputum. We report that the percentage of BONCAT-labeled (i.e. active) bacterial cells varies substantially between subjects (6-56%). We use fluorescence-activated cell sorting (FACS) and genomic sequencing to assign taxonomy to BONCAT-labeled cells. While many abundant taxa are indeed active, most bacterial species detected by conventional molecular profiling show a mixed population of both BONCAT-labeled and unlabeled cells, suggesting heterogeneous growth rates in sputum. Differentiating translationally active subpopulations adds to our evolving understanding of CF lung disease and may help guide antibiotic therapies targeting bacteria most likely to be susceptible.
对囊性纤维化肺部微生物组的非培养研究，为多微生物群落在疾病中的基础作用提供的机制见解甚少。要弄清各个分类群对 CF 发病机制的具体贡献，需要全面了解它们在感染部位的生态生理学。我们假设只有 CF 微生物组的一个子集具有翻译活性，并且这些活性在不同的个体之间存在差异。在这里，我们应用生物正交非典型氨基酸标记（BONCAT）来可视化和定量分析咳出的痰中的细菌翻译活性。我们报告说，BONCAT 标记（即活跃）的细菌细胞的百分比在个体之间差异很大（6-56%）。我们使用荧光激活细胞分选（FACS）和基因组测序将 BONCAT 标记的细胞分配给分类。虽然许多丰富的分类群确实是活跃的，但通过常规分子分析检测到的大多数细菌物种都显示出 BONCAT 标记和未标记细胞的混合群体，这表明在痰中存在异速生长。区分翻译活跃的亚群增加了我们对 CF 肺部疾病的不断发展的理解，并可能有助于指导针对最有可能敏感的细菌的抗生素治疗。
