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Intracellular Persistence of Pseudomonas Aeruginosa in Airway Epithelial Cells and in Cystic Fibrosis Lung

Faure, E. and Jorth, P. and Berube, J. and Brochiero, E. and Newman, D. and Rousseau, S. and Nguyen, D. (2018) Intracellular Persistence of Pseudomonas Aeruginosa in Airway Epithelial Cells and in Cystic Fibrosis Lung. American Journal of Respiratory and Critical Care Medicine, 197 . Art. No. A6262. ISSN 1073-449X.

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Rationale: Cystic Fibrosis (CF) lung disease is characterized by chronic Pseudomonas aeruginosa infections in airways. Why P. aeruginosa persists despite antimicrobial and activation of immune responses remains incompletely understood. To date, the prevailing view suggests that impaired muco-cilliary clearance and biofilm lifestyle are the primary mechanisms contributing to the persistence in CF lung. Although P. aeruginosa is an extracellular pathogen, previous in vitro studies have reported that it is internalized in epithelial cells. This led us to hypothesize that P. aeruginosa is internalized and persists in lung epithelial cells, contributing to P. aeruginosa's evasion of extracellular antibacterials and host defenses. Methods: We have developed an in vitro model of long-term intracellular persistence of P. aeruginosa in human airway epithelial cells, using several cell lines (Beas-2B, CBFE and CFBEΔF508), and have tested different P. aeruginosa strains (PAO1, CHA, isogenic mutants). Epithelial cells, grown as immersed monolayers, or polarized at the air-liquid interface, and incubated with P. aeruginosa at MOl 1 during 4 hours. For the following 5 days, epithelial cells are treated with tobramycin to maintain the extracellular culture milieu sterile. Intracellular bacterial burden was measured at different time points by plate counting of CFU. We assessed cell death by LOH assay. Confocal microscopy was used to localize and count intracellular P. aeruginosa. Finally, we use MiPACT, (Microbial identification after Passive CLARITY Technique), a technique that allows direct visualization of P. aeruginosa, using targeted antibody or RNA probe, in cleared lungs from CF patient, creating a three-dimensional biogeographical map of the lung tissue and the infecting pathogens. Results: We showed that P. aeruginosa persists and replicates in B2B and CFBE cells over 5 days without causing significant host cell cytotoxicity. Approximately 15% of initial bacterial inoculum is internalized, and the intracellular bacterial burden increases 2-fold over 5 days. Moreover, bacterial persistence is increased ~2-fold in CFBEΔF508 cells compared to CFBE. Non-flagellated mutant was showed increased persistence. Additionally, we showed that P. aeruginosa co-localizes with LAMP-1. Using MiPACT, we showed a positive intracellular staining of P. aeruginosa in airway epithelial cells from CF lung explants. Conclusion: Our in vitro model suggests that P. aeruginosa is readily internalized and persists more in CF airway epithelial cell. The possibility that P. aeruginosa may have an intracellular niche within the CF lung highlights a potentially new mechanism of bacterial persistence in CF lung disease. This abstract is funded by: None

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Jorth, P.0000-0002-0981-740X
Newman, D.0000-0003-1647-1918
Additional Information:© 2018 American Thoracic Society.
Record Number:CaltechAUTHORS:20181206-104542777
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91534
Deposited By: Tony Diaz
Deposited On:06 Dec 2018 19:16
Last Modified:09 Mar 2020 13:18

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