Abstract
Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. β-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by β-lactams have been hampered by lack of access to a suitable substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate the utility of this strategy by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2 (PBP2), which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different β-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.
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Acknowledgements
The authors thank J.X. Wang at Harvard Small Molecule Mass Spectrometry Facility for assistance in running LC–MS and LC–MS/MS and interpretation of MS/MS spectra. This work was funded by US National Institutes of Health grants R01 GM100951 to N.R., R01 GM076710 to S.W. and D.K., R01 GM066174 to D.K., and a Singapore A*STAR NSS (PhD) scholarship to Y.Q.
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Y.Q. and V.S. developed methods to isolate and quantify Lipid II from Staphylococcus aureus, Escherichia coli and Bacillus subtilis, based in part on studies conducted by F.R. and K.S.; Y.Q. and V.S. purified S. aureus PBP2; Y.Q. performed studies on PBP2 transpeptidase activity and characterized β-lactam inhibition; N.R. constructed the E. coli MurJA29C strain; S.W. and D.K. designed and supervised the project. The manuscript and figures were prepared by Y.Q., V.S., S.W., and D.K. with input from all authors.
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Qiao, Y., Srisuknimit, V., Rubino, F. et al. Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams. Nat Chem Biol 13, 793–798 (2017). https://doi.org/10.1038/nchembio.2388
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DOI: https://doi.org/10.1038/nchembio.2388
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