Abstract
The human gut microbiome modulates physiological functions and pathologies; however, a mechanistic understanding of microbe–host and microbe–microbe interactions remains elusive owing to a lack of suitable approaches to monitor obligate anaerobic bacterial populations. Common genetically encoded fluorescent protein reporters, derived from the green fluorescent protein, require an oxidation step for fluorescent light emission and therefore are not suitable for use in anaerobic microbes residing in the intestine. Fluorescence in situ hybridization is a useful alternative to visualize bacterial communities in their natural niche; however, it requires tissue fixation. We therefore developed an approach for the real-time detection and monitoring of live communities of anaerobic gut commensals in their natural environment. We leverage the bacterial cells’ reliance on sugars for macromolecule synthesis in combinatorial click chemistry labeling, where the addition of azide-modified sugars to the culturing media enables the fluorescence labeling of newly synthesized molecules via the addition of combinations of exogenous fluorophores conjugated to cyclooctynes. This process is suitable for labeling communities of live anaerobic gut bacteria with combinations of fluorophores that do not require oxygen to mature and fluoresce, and that can be detected over time in their natural environments. The labeling procedure requires 4–9 d, depending on the varying growth rates of different bacterial strains, and an additional 1–2 d for the detection and monitoring steps. The protocol can be completed by users with basic expertise in bacterial culturing.
Key points
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A combinatorial click chemistry approach that enables the incorporation of combinations of fluorescently labeled cyclooctynes to newly synthesized molecules in anaerobic bacteria via the addition of azide-modified sugars to the culturing media.
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Alternative strategies for labeling and tracking bacteria include genetically expressed fluorescent proteins, fluorescence in situ hybridization, bilin-binding fluorescent proteins and fluorescent D-amino acids labeling.
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Acknowledgements
We thank the Geva-Zatorsky laboratory members, especially R. Naddaf and A. Landers for fruitful discussions and contributions. We also thank A. Grau, M. Holdengreber and the biomedical core facility at the Technion Rappaport faculty of Medicine for their help with flow cytometry and confocal microscopy. This work was supported by the Technion Institute of Technology, ‘Keren Hanasi’, Cathedra, Rappaport Technion Integrated Cancer Center, the Alon Fellowship for Outstanding Young Researchers, the Israeli Science Foundation (grant 3165/20), D. Dan and Betty Kahn Foundation, the Seerave Foundation, the German–Israeli Foundation for Scientific Research and Development (grant I-1076-416.6-20), CIFAR (Azrieli Global Scholars; grant FL-000969), the Israel Cancer Research Fund (grant 1016142), Human Frontier Science Program Career Development Award (grant CDA00025/2019-C), Johnson & Johnson WiSTEM2D (grant 1015773), the Gutwirth Foundation Award and the European Union (ERC, ExtractABact, 101078712). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. N.G.-Z. is an Azrieli Global Scholar at the Canadian Institute for Advanced Research, a Johnson & Johnson WiSTEM2D scholar and a Horev Fellow (Taub Foundation). H.H. was supported by a Leonard and Diane Sherman Interdisciplinary Graduate School fellowship, the Wjuniski Fellowship Fund for the MD/PhD Medical Scientist Program and a VATAT fellowship for outstanding doctoral students from the Arab community. N.B. was supported in part at the Technion by the Israel Council for Higher Education Council under a PBC fellowship. Figures were created with the assistance of Y. Abraham.
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Key references using this protocol
Geva-Zatorsky, N. et al. Nat. Med. 21, 1091–1100 (2015): https://doi.org/10.1038/nm.3929
Hajjo. H. et al. Front. Microbiol. 12, 750624 (2021): https://doi.org/10.3389/fmicb.2021.750624
Extended data
Extended Data Fig. 1 Assessment of the nonspecific binding of cyclooctyne fluorophores using confocal microscopy.
To assess the nonspecific binding of cyclooctyne fluorophores to bacteria, B. fragilis was grown in basal media (BM) or GalNAz-supplemented basal media (GalNAz) and then incubated with AZDye647, AZDye488 and TAMRA cyclooctyne fluorophores. The fluorescence was assessed by confocal microscopy imaging. Gamma and range correction techniques were identically applied to both the images and their respective controls.
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Hajjo, H., Bhardwaj, N., Gefen, T. et al. Combinatorial fluorescent labeling of live anaerobic bacteria via the incorporation of azide-modified sugars into newly synthesized macromolecules. Nat Protoc 18, 3767–3786 (2023). https://doi.org/10.1038/s41596-023-00896-7
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DOI: https://doi.org/10.1038/s41596-023-00896-7
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