Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs
Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.
MS/MS spectra have been provided in Supplementary data file.The software developments required to predict and analyze the labeled/unlabeled muropeptide ions isotopic clusters either in MS or MS/MS experiments are hosted at https://gitlab.com/kantundpeterpan/masseltof and published under a Free Software license.
Article and author information
Agence Nationale de la Recherche (ANR-16-CE11-0030-12)
- Heiner Atze
- Michel Arthur
National Institute of Allergy and Infectious Diseases (R56AI045626)
- Yucheng Liang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bavesh D Kana, University of the Witwatersrand, South Africa
- Preprint posted: August 6, 2021 (view preprint)
- Received: August 6, 2021
- Accepted: June 9, 2022
- Accepted Manuscript published: June 9, 2022 (version 1)
- Version of Record published: July 1, 2022 (version 2)
© 2022, Atze et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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