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. 2019 Dec;11(4):e74.
doi: 10.1002/cpch.74.

Metabolic Incorporation of N-Acetyl Muramic Acid Probes into Bacterial Peptidoglycan

Kristen E DeMeester  1 , Hai Liang  1   2 , Junhui Zhou  1 , Kimberly A Wodzanowski  1 , Benjamin L Prather  1 , Cintia C Santiago  1   3 , Catherine L Grimes  1   4
Affiliations

Metabolic Incorporation of N-Acetyl Muramic Acid Probes into Bacterial Peptidoglycan

Kristen E DeMeester et al. Curr Protoc Chem Biol. 2019 Dec.

Abstract

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.

Keywords: bacterial peptidoglycan; bioorthogonal chemistry; carbohydrates; click chemistry; fluorescent labeling; mass spectrometry; metabolic incorporation; microscopy.

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Figures

Figure 1.
Figure 1.. Chemical structure of bacterial PG of both Gram-negative and Gram-positive organisms.
Gram-negative bacterial cells normally use meso-diaminopimelic acid (meso-DAP, red) and d-glutamic acid (d-Glu, red) in their PG structure. In Gram-positive bacteria, the peptide chains normally consist of d-isoglutamine (d-isoGln, green) and l-lysine (l-Lys, green)
Figure 2.
Figure 2.. PG biosynthesis begins with the formation of UDP-NAM through MurA/B and UDP-NAG.
Recycling enzymes AmgK/MurU provide another route to synthesize UDP-NAM with NAM as the building block. UDP-NAM is converted into Park’s nucleotide through enzymes MurC-F. MraY links Park’s nucleotide to the cell membrane where MurG then glycosylates this Lipid I fragment to form Lipid II. MurJ transports Lipid II into the periplasmic space where transglycosylases (TGase) and transpeptidases (TPase) further cross-link these molecules to form the mature PG. NAM probes (blue) with biorthogonal functionality either at the 2-N position (X) or 3-lactic acid position (Y) are metabolically incorporated into PG through both recycling and biosynthetic machineries.
Figure 3.
Figure 3.. Chemical Modifications of Bacterial PG.
Summary of PG chemical modifications with fluorophores to the peptide and carbohydrate portions of the PG polymer are highlighted in different colors.
Figure 4.:
Figure 4.:. Synthesis of Peracetylated 2-Azido Glucosamine (1).
Figure 5.:
Figure 5.:. Synthesis of 2N- functionalized NAM derivatives.
Figure 6.
Figure 6.. Synthesis of 3-azido NAM methyl ester.
Figure 7.
Figure 7.
A) 1HNM spectra of 3-azido NAM methyl ester: 1H NMR (400 MHz, Methanol-d4) δ 5.33 (d, J = 2.3 Hz, 1H, α-H1), 4.91 – 4.88 (m, 1H, α-CHCH2CH3), 4.83 (dd, J = 5.2, 3.8 Hz, β-CHCH2CH3), 4.66 – 4.59 (m, β-H1, anomeric ratio α/β=2.57), 3.83 (s, 3H, α-CH3O), 3.80 (s, β-CH3O), 3.77 – 3.65 (m, α and β-C6, α-C2, α-C4 and α-C3), 3.62–3.47(m, α and β-CHCH2N3, β-C2, β-C4, β-C5 and β-C3), 3.31–3.27 (m, α-C5), 2.04 (double singlet, α and β-CH3C(O)NH); B)13C NMR spectra: 13C NMR (101 MHz, Methanol-d4) δ 173.29, 172.36, 172.10, 171.60 (carbonyls), 95.91 (β-C1), 90.51 (α-C1), 81.53 (β-C3), 79.14 (α-CHCH2N3), 78.83 (β-CHCH2N3), 78.35 (α-C3), 76.48 (α-C5), 72.02 (β-C5), 71.42 (α-C4), 71.06 (β-C4), 61.00 (β-C6), 60.90 (α-C6), 56.43 (β-C2), 54.21 (α-C2, 52.47 (α and β-CHCH2N3), 51.72 (α-CH3O), 51.54 (β-CH3O), 21.72 (β-CH3C(O)NH), 21.48 (α-CH3C(O)NH).
Figure 8.
Figure 8.
Structured illumination microscopy images of cells treated with 2 AzNAM filtered with Waters preparative HPLC/MS or with Maxi-Clean™ SPE C18 plug and clicked with AF-Alk488 (white) (scale bars, 5 μM). Images are representative of a minimum of five fields of view per replicate with at least three technical and biological replicates.
Figure 9.
Figure 9.. Mass spectrometry verification of 3AzNAM incorporation into bacterial PG by lysozyme digestion.
Overall experimental strategy to identify fluorescent PG fragments in E. coli ΔMurQ KU cells remodeled with 3AzNAM methyl ester. Two of the potential lysozyme fragments from the digestion are shown as A and B. Mass spectrometry results are shown for fragment A. Peaks are confirmed by observing the proper isotope pattern with the observed mass within +/− 10 ppm of the expected mass over two technical replicates of 2 biological replicates.
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