Metabolic Incorporation of N-Acetyl Muramic Acid Probes into Bacterial Peptidoglycan.
bacterial peptidoglycan
bioorthogonal chemistry
carbohydrates
click chemistry
fluorescent labeling
mass spectrometry
metabolic incorporation
microscopy
Journal
Current protocols in chemical biology
ISSN: 2160-4762
Titre abrégé: Curr Protoc Chem Biol
Pays: United States
ID NLM: 101559235
Informations de publication
Date de publication:
12 2019
12 2019
Historique:
entrez:
26
11
2019
pubmed:
26
11
2019
medline:
15
7
2020
Statut:
ppublish
Résumé
Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh-like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N-acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper-catalyzed azide-alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super-resolution microscopy and high-resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user-friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of peracetylated 2-azido glucosamine Basic Protocol 2: Synthesis of 2-azido and 2-alkyne NAM Basic Protocol 3: Synthesis of 3-azido NAM methyl ester Basic Protocol 4: Incorporation of NAM probes into bacterial peptidoglycan Basic Protocol 5: Confirmation of bacterial cell wall remodeling by mass spectrometry.
Identifiants
pubmed: 31763799
doi: 10.1002/cpch.74
pmc: PMC7591266
mid: NIHMS1067485
doi:
Substances chimiques
Alkynes
0
Azides
0
Fluorescent Dyes
0
Muramic Acids
0
Peptidoglycan
0
N-acetylmuramic acid
246FXU111L
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e74Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM133395
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM008550
Pays : United States
Organisme : NIGMS NIH HHS
ID : P30 GM110758
Pays : United States
Organisme : NCI NIH HHS
ID : U01 CA221230
Pays : United States
Organisme : NIGMS NIH HHS
ID : P20 GM103446
Pays : United States
Organisme : NIGMS NIH HHS
ID : P20 GM104316
Pays : United States
Informations de copyright
© 2019 John Wiley & Sons, Inc.
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