Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs
- INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, France
- Sorbonne Université-INSERM, France
- PAPPSO, Universite Paris-Saclay, INRAE, CNRS, France
Abstract
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.
Data availability
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
Author details
Funding
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.
Reviewing Editor
- Bavesh D Kana, University of the Witwatersrand, South Africa
Publication history
- 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)
Copyright
© 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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Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs
eLife 11:e72863.
https://doi.org/10.7554/eLife.72863
Further reading
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Metazoans establish mutually beneficial interactions with their resident microorganisms. However, our understanding of the microbial cues contributing to host physiology remains elusive. Previously, we identified a bacterial machinery encoded by the dlt operon involved in Drosophila melanogaster’s juvenile growth promotion by Lactiplantibacillus plantarum. Here, using crystallography combined with biochemical and cellular approaches, we investigate the physiological role of an uncharacterized protein (DltE) encoded by this operon. We show that lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarumNC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarumNC8 and Drosophila melanogaster as a symbiosis model, we establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. Our results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.
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Elucidating the role of one of the proteins produced by Lactiplantibacillus plantarum reveals a new molecule that allows this gut bacterium to support the development of fruit fly larvae.
