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.
Bibliographical noteFunding Information:
We thank Roland Hatzenpichler (Montana State University) for technical advice, Alex Vitti for figure design, the care team at the UMN Adult CF Treatment Center and their patients for participating in the research. This work was supported by a Gilead Sciences Investigator Sponsored Research Award and a Cystic Fibrosis Foundation Research Grant (HUNTER16G0) to R.C.H. K.A.B. was supported by a NIH Lung Sciences T32 fellowship (#2T32HL007741-21) awarded through the National Heart, Lung, and Blood Institute. S.K.L. received support from T32 (#T90 DE0227232) and F31 (#F31 DE027602) fellowships through the National Institute of Dental and Craniofacial Research.
© 2020, The Author(s).
- Amino Acids/metabolism
- Cystic Fibrosis/microbiology
- Protein Biosynthesis
- Pseudomonas aeruginosa/physiology
PubMed: MeSH publication types
- Research Support, Non-U.S. Gov't
- Journal Article
- Research Support, N.I.H., Extramural