Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling

Abstract

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Investigating Peptidoglycan Recycling Pathways inTannerella forsythiawithN-Acetylmuramic Acid Bioorthogonal Probes

“…In the case of the newly developed DH5α-Tf-KU strain, when supplementing with AzNAM, we found that DH5α-Tf-KU was able to recover cell growth at all AzNAM concentrations tested for approximately 3 h and concentrations of 150 μM and above for at least 6 h (Figure A). These conditions are superior to the previous conditions for EQKU cells which could only recover growth with AzNAM at concentrations of 600 μM and above (Figure B) . These data suggest that the AmgK/MurU homologues from T. forsythia are more efficient at utilizing the NAM probes and are therefore better suited for a PG remodeling strategy.…”

“…These conditions are superior to the previous conditions for EQKU cells which could only recover growth with AzNAM at concentrations of 600 μM and above (Figure 3B). 18 These data suggest that the AmgK/MurU homologues from T. forsythia are more efficient at utilizing the NAM probes and are therefore better suited for a PG remodeling strategy.…”

“…We then utilized a plate reader assay to perform a concentration screen to determine the minimal concentration of AzNAM needed to recover the growth of DH5α-Tf-KU cells, as AzNAM is an unnatural NAM substrate that is known to reduce P. putida AmgK substrate turnover rate by 10-fold . Previously for labeling experiments, we have employed a different strain of E. coli , E. coli Δ MurQ-KU (EQKU), which utilizes AmgK and MurU enzymes from P. putida for recycling NAM substrates . Although this strain worked well in initial remodeling and fluorescent imaging experiments, it was limited in how effective it could use low concentrations of unnatural NAM substrates like AzNAM for sustaining growth and labeling experiments.…”

“…15 Previously for labeling experiments, we have employed a different strain of E. coli, E. coli ΔMurQ-KU (EQKU), which utilizes AmgK and MurU enzymes from P. putida for recycling NAM substrates. 18 Although this strain worked well in initial remodeling and fluorescent imaging experiments, it was limited in how effective it could use low concentrations of unnatural NAM substrates like AzNAM for sustaining growth and labeling experiments. In the case of the newly developed DH5α-Tf-KU strain, when supplementing with AzNAM, we found that DH5α-Tf-KU was able to recover cell growth at all AzNAM concentrations tested for approximately 3 h and concentrations of 150 μM and above for at least 6 h (Figure 3A).…”

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria

“…30 μl of DBCO-488 was added to appropriate wells. The plate was incubated at 37°C in the plate reader where it was shaken for 2 min, scanned at 600 nm, and then repeated every 20 min for 6 h ( Brown et al, 2021a ). Cell growth curves were formulated with Origin 2019.…”