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. Author manuscript; available in PMC: 2023 Feb 9.
Published in final edited form as: Chem Rev. 2021 Dec 14;122(3):3336–3413. doi: 10.1021/acs.chemrev.1c00729

Figure 9.

Figure 9.

Examples of in vitro and in vivo D-amino acid reporter labeling of PG. (A) Metabolic incorporation of HADA (17) into E. coli, B. subtilis, and A. tumefaciens. Bacteria were incubated in HADA, fixed, and imaged directly (top panel) or subjected to sacculus isolation (i.e., intact PG isolation) and then imaged (bottom panel). WGA595 is a red-fluorescent lectin that binds to PG glycan strands. Reproduced with permission from ref 76. Copyright 2012 Wiley-VCH. (B) Metabolic incorporation of AlkDA (10) into E. coli (Ec), L. monocytogenes (Lm), C. glutamicum (Cg), and M. tuberculosis (Mt). Bacteria were incubated in either D-Ala (control, top panel) or AlkDA (bottom panel), fixed, subjected to CuAAC with azido-488, and imaged. (C) Imaging of L. monocytogenes-infected macrophages with AlkDA (10). Macrophages were infected with red fluorescent protein (RFP)-expressing L. monocytogenes and extracellular bacteria were removed, then incubated in AlkDA (10), fixed, subjected to CuAAC with azido-488, and imaged. (B) and (C) were reproduced with permission from ref 77 (https://pubs.acs.org/doi/full/10.1021/ja505668f). Copyright 2013 American Chemical Society. Further permission related to the material excerpted should be directed to the American Chemical Society. (D) Sequential labeling of S. venezuelae with variable-color fluorescent D-amino acid derivatives. Bacteria were sequentially incubated for short pulses with different dye-conjugated D-amino acids possessing orthogonal excitation/emission wavelengths (structures not shown in Figure 7), fixed, and imaged. Arrows indicate new branching sites of the cell wall. Reproduced from ref 109 with permission from the Royal Society of Chemistry. (E) Ex vivo and in vivo labeling of S. aureus PG in host C. elegans using the tetrazine reagent TetDAC (25, Figure 10A). C. elegans was infected with green fluorescent protein (GFP)-expressing S. aureus that had been (i) pre-treated with TetDAC (25) for ex vivo labeling or (ii) not pre-treated for in vivo labeling. C. elegans was washed to remove uncolonized bacteria, then (i) reacted with TCO-Cy5 fluorophore and imaged for ex vivo labeling or (ii) treated with TetDAC (25), reacted with TCO-Cy5 fluorophore, and imaged for in vivo labeling. Reproduced with permission from ref 80. Copyright 2017 American Chemical Society.