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Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

Nature Chemical Biology volume 13, pages 793–798 (2017)Cite this article

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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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Figure 1: Biosynthetic pathway for peptidoglycan assembly in Staphylococcus aureus.
Figure 2: Lipid II can be accumulated in bacteria using chemical probes that block Lipid II export or polymerization.
Figure 3: Large quantities of native S. aureus Lipid II can be isolated with good purity by extraction.
Figure 4: The synthesis of crosslinked peptidoglycan was reconstituted using native S. aureus Lipid II.
Figure 5: Direct transpeptidase activity assay enables comparison of β-lactam inhibition of S. aureus PBP2.

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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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  1. Yuan Qiao and Veerasak Srisuknimit: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Harvard Medical School, Boston, Massachusetts, USA

    Yuan Qiao, Kaitlin Schaefer & Suzanne Walker

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Yuan Qiao, Veerasak Srisuknimit, Frederick Rubino, Kaitlin Schaefer & Daniel Kahne

  3. Department of Microbiology, Ohio State University, Columbus, Ohio, USA

    Natividad Ruiz

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Contributions

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.

Corresponding authors

Correspondence to Suzanne Walker or Daniel Kahne.

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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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