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Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform

The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded flu...

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Autores principales: Anderson, David M., Logan, Matthew G., Patty, Sara S., Kendall, Alexander J., Borland, Christina Z., Pfeifer, Carmem S., Kreth, Jens, Merritt, Justin L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10592883/
https://www.ncbi.nlm.nih.gov/pubmed/37873282
http://dx.doi.org/10.1101/2023.10.02.560575
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author Anderson, David M.
Logan, Matthew G.
Patty, Sara S.
Kendall, Alexander J.
Borland, Christina Z.
Pfeifer, Carmem S.
Kreth, Jens
Merritt, Justin L.
author_facet Anderson, David M.
Logan, Matthew G.
Patty, Sara S.
Kendall, Alexander J.
Borland, Christina Z.
Pfeifer, Carmem S.
Kreth, Jens
Merritt, Justin L.
author_sort Anderson, David M.
collection PubMed
description The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, (PL)Blaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto (PL)Blaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to (PL)Blaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.
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spelling pubmed-105928832023-10-24 Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform Anderson, David M. Logan, Matthew G. Patty, Sara S. Kendall, Alexander J. Borland, Christina Z. Pfeifer, Carmem S. Kreth, Jens Merritt, Justin L. bioRxiv Article The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, (PL)Blaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto (PL)Blaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to (PL)Blaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability. Cold Spring Harbor Laboratory 2023-10-02 /pmc/articles/PMC10592883/ /pubmed/37873282 http://dx.doi.org/10.1101/2023.10.02.560575 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Anderson, David M.
Logan, Matthew G.
Patty, Sara S.
Kendall, Alexander J.
Borland, Christina Z.
Pfeifer, Carmem S.
Kreth, Jens
Merritt, Justin L.
Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title_full Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title_fullStr Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title_full_unstemmed Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title_short Microbiome imaging goes à la carte: Incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (FAST) imaging platform
title_sort microbiome imaging goes à la carte: incorporating click chemistry into the fluorescence-activating and absorption-shifting tag (fast) imaging platform
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10592883/
https://www.ncbi.nlm.nih.gov/pubmed/37873282
http://dx.doi.org/10.1101/2023.10.02.560575
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