Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024:2727:1-16.
doi: 10.1007/978-1-0716-3491-2_1.

Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall

Stephen N Hyland  1 , Sreedevi Chinthamani  2 , Sushanta Ratna  1 , Kimberly A Wodzanowski  1 , Liam-Michael D Sandles  1 , Kiyonobu Honma  2 , Catherine Leimkuhler-Grimes  3   4 , Ashu Sharma  5
Affiliations

Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall

Stephen N Hyland et al. Methods Mol Biol. 2024.

Abstract

The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through "click chemistry" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes.

Keywords: Bioorthogonal; Cell wall recycling; Click chemistry; Mass spectrometry; Microscopy; Peptidoglycan; Tannerella forsythia.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Representative images of T. forsythia labeling. T. forsythia cells are fluorescently visualized following CuAAC click reaction with Cy5-Alkyne. NAM cells do not have the azide handle and therefore do not react with the fluorophores, leading to no fluorescence. Scale bars = 20 μm. (Reprinted with permission from Wodzanowski et al. [7]. Copyright 2023 American Chemical Society)
Fig. 2
Fig. 2
Representative images of CuAAC labeling of DH5α-Tf-KU cell wall. Top row) AzNAM. Bottom row) NAM control. Confocal images were taken on a Zeiss LSM 800 microscope using Zen Blue. Alk-488 was visualized using the green laser line. BF = brightfield. Alk-488 = Alexa Fluor 488 Alkyne. Scale bars = 10 μm
Fig. 3
Fig. 3
Representative images of SPAAC labeling of DH5α-Tf-KU cell wall. Top row) AzNAM. Bottom row) NAM control. Confocal images were taken on a Zeiss LSM 800 microscope using Zen Blue. DBCO-488 was visualized using the green laser line. BF = brightfield. DBCO-488 = AZDye™ 488 DBCO. Scale bars = 10 μm
Fig. 4
Fig. 4
Representative mass spectrometry results for NAM incorporation. (a) (Top row) Total ion chromatogram. (Bottom row) Mass range filter indicating relative abundance for corresponding NAM disaccharide mass. (b) (Top row) NAM sample isotopic peak distribution with corresponding structure of interest. (Bottom row) Simulated isotopic peak distribution using molecular formula of the structure of interest
Fig. 5
Fig. 5
Representative mass spectrometry results for AzNAM incorporation. (a) (Top row) Total ion chromatogram. (Bottom row) Mass range filter indicating relative abundance for corresponding AzNAM disaccharide mass. (b) (Top row) AzNAM sample isotopic peak distribution with corresponding structure of interest. (Bottom row) Simulated isotopic peak distribution using molecular formula of the structure of interest
See this image and copyright information in PMC

References

    1. Kilian M, Chapple I, Hannig M et al. (2016) The oral microbiome – an update for oral healthcare professionals. Br Dent J 221:657–666 - PubMed
    1. Kleinstein S (2020) E et al. inflammatory networks linking oral microbiome with systemic health and disease. J Dent Res 99(10): 1131–1139 - PMC - PubMed
    1. Tanner AC, Izard J (2006) Tannerella forsythia, a periodontal pathogen entering the genomic era. Periodontol 2000 42:88–113 - PubMed
    1. Ruscitto A et al. (2016) Identification of a novel N-acetylmuramic acid transporter in Tannerella forsythia. J Bacteriol 198(22):3119–3125 - PMC - PubMed
    1. Gisin J et al. (2013) A cell wall recycling shortcut that bypasses peptidoglycan de novo biosynthesis. Nat Chem Biol 9(8):491–493 - PubMed

Publication types

LinkOut - more resources