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Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa

Pseudomonas aeruginosa thrives in the airways of individuals with cystic fibrosis, in part by forming robust biofilms that are resistant to immune clearance or antibiotic treatment. In the cystic fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating with the formatio...

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Autores principales: Tchagang, Caetanie F., Mah, Thien-Fah, Campbell-Valois, François-Xavier
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10623331/
https://www.ncbi.nlm.nih.gov/pubmed/37928662
http://dx.doi.org/10.3389/fmicb.2023.1245755
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author Tchagang, Caetanie F.
Mah, Thien-Fah
Campbell-Valois, François-Xavier
author_facet Tchagang, Caetanie F.
Mah, Thien-Fah
Campbell-Valois, François-Xavier
author_sort Tchagang, Caetanie F.
collection PubMed
description Pseudomonas aeruginosa thrives in the airways of individuals with cystic fibrosis, in part by forming robust biofilms that are resistant to immune clearance or antibiotic treatment. In the cystic fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating with the formation of anoxic pockets. In this environment, P. aeruginosa can use nitrate instead of oxygen to grow. Current fluorescent reporters for studying P. aeruginosa are limited to the GFP and related analogs. However, these reporters require oxygen for the maturation of their chromophore, making them unsuitable for the study of anaerobically grown P. aeruginosa. To overcome this limitation, we evaluated seven alternative fluorescent proteins, including iLOV, phiLOV2.1, evoglow-Bs2, LucY, UnaG, Fluorescence-Activating and Absorption-Shifting Tag (FAST), and iRFP670, which have been reported to emit light under oxygen-limiting conditions. We generated a series of plasmids encoding these proteins and validated their fluorescence using plate reader assays and confocal microscopy. Six of these proteins successfully labeled P. aeruginosa in anoxia. In particular, phiLOV2.1 and FAST provided superior fluorescence stability and enabled dual-color imaging of both planktonic and biofilm cultures. This study provides a set of fluorescent reporters for monitoring P. aeruginosa under low-oxygen conditions. These reporters will facilitate studies of P. aeruginosa in biofilms or other contexts relevant to its pathogenesis, such as those found in cystic fibrosis airways. Due to the broad host range of our expression vector, the phiLOV2.1 and FAST-based reporters may be applicable to the study of other Gram-negative bacteria that inhabit similar low-oxygen niches.
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spelling pubmed-106233312023-11-04 Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa Tchagang, Caetanie F. Mah, Thien-Fah Campbell-Valois, François-Xavier Front Microbiol Microbiology Pseudomonas aeruginosa thrives in the airways of individuals with cystic fibrosis, in part by forming robust biofilms that are resistant to immune clearance or antibiotic treatment. In the cystic fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating with the formation of anoxic pockets. In this environment, P. aeruginosa can use nitrate instead of oxygen to grow. Current fluorescent reporters for studying P. aeruginosa are limited to the GFP and related analogs. However, these reporters require oxygen for the maturation of their chromophore, making them unsuitable for the study of anaerobically grown P. aeruginosa. To overcome this limitation, we evaluated seven alternative fluorescent proteins, including iLOV, phiLOV2.1, evoglow-Bs2, LucY, UnaG, Fluorescence-Activating and Absorption-Shifting Tag (FAST), and iRFP670, which have been reported to emit light under oxygen-limiting conditions. We generated a series of plasmids encoding these proteins and validated their fluorescence using plate reader assays and confocal microscopy. Six of these proteins successfully labeled P. aeruginosa in anoxia. In particular, phiLOV2.1 and FAST provided superior fluorescence stability and enabled dual-color imaging of both planktonic and biofilm cultures. This study provides a set of fluorescent reporters for monitoring P. aeruginosa under low-oxygen conditions. These reporters will facilitate studies of P. aeruginosa in biofilms or other contexts relevant to its pathogenesis, such as those found in cystic fibrosis airways. Due to the broad host range of our expression vector, the phiLOV2.1 and FAST-based reporters may be applicable to the study of other Gram-negative bacteria that inhabit similar low-oxygen niches. Frontiers Media S.A. 2023-10-20 /pmc/articles/PMC10623331/ /pubmed/37928662 http://dx.doi.org/10.3389/fmicb.2023.1245755 Text en Copyright © 2023 Tchagang, Mah and Campbell-Valois. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Tchagang, Caetanie F.
Mah, Thien-Fah
Campbell-Valois, François-Xavier
Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title_full Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title_fullStr Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title_full_unstemmed Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title_short Anaerobic fluorescent reporters for live imaging of Pseudomonas aeruginosa
title_sort anaerobic fluorescent reporters for live imaging of pseudomonas aeruginosa
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10623331/
https://www.ncbi.nlm.nih.gov/pubmed/37928662
http://dx.doi.org/10.3389/fmicb.2023.1245755
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