Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

  1. Heiner Atze
  2. Yucheng Liang
  3. Jean-Emmanuel Hugonnet
  4. Arnaud Gutierrez
  5. Filippo Rusconi  Is a corresponding author
  6. Michel Arthur  Is a corresponding author
  1. INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, France
  2. Sorbonne Université-INSERM, France
  3. 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

  1. Heiner Atze

    INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1497-6373
  2. Yucheng Liang

    Sorbonne Université-INSERM, PARIS, France
    Competing interests
    The authors declare that no competing interests exist.
  3. Jean-Emmanuel Hugonnet

    INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  4. Arnaud Gutierrez

    INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  5. Filippo Rusconi

    PAPPSO, Universite Paris-Saclay, INRAE, CNRS, Paris, France
    For correspondence
    filippo.rusconi@universite-paris-saclay.fr
    Competing interests
    The authors declare that no competing interests exist.
  6. Michel Arthur

    INSERM, UMR-S 1138, Centre de Recherche des Cordeliers, Paris, France
    For correspondence
    michel.arthur@crc.jussieu.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1007-636X

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

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Version history

  1. Preprint posted: August 6, 2021 (view preprint)
  2. Received: August 6, 2021
  3. Accepted: June 9, 2022
  4. Accepted Manuscript published: June 9, 2022 (version 1)
  5. 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.

Metrics

  • 1,172
    views
  • 341
    downloads
  • 8
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Heiner Atze
  2. Yucheng Liang
  3. Jean-Emmanuel Hugonnet
  4. Arnaud Gutierrez
  5. Filippo Rusconi
  6. Michel Arthur
(2022)
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

Share this article

https://doi.org/10.7554/eLife.72863

Further reading

    1. Microbiology and Infectious Disease
    Brian G Vassallo, Noemie Scheidel ... Dennis H Kim
    Research Article

    The microbiota is a key determinant of the physiology and immunity of animal hosts. The factors governing the transmissibility of viruses between susceptible hosts are incompletely understood. Bacteria serve as food for Caenorhabditis elegans and represent an integral part of the natural environment of C. elegans. We determined the effects of bacteria isolated with C. elegans from its natural environment on the transmission of Orsay virus in C. elegans using quantitative virus transmission and host susceptibility assays. We observed that Ochrobactrum species promoted Orsay virus transmission, whereas Pseudomonas lurida MYb11 attenuated virus transmission relative to the standard laboratory bacterial food Escherichia coli OP50. We found that pathogenic Pseudomonas aeruginosa strains PA01 and PA14 further attenuated virus transmission. We determined that the amount of Orsay virus required to infect 50% of a C. elegans population on P. lurida MYb11 compared with Ochrobactrum vermis MYb71 was dramatically increased, over three orders of magnitude. Host susceptibility was attenuated even further in the presence of P. aeruginosa PA14. Genetic analysis of the determinants of P. aeruginosa required for attenuation of C. elegans susceptibility to Orsay virus infection revealed a role for regulators of quorum sensing. Our data suggest that distinct constituents of the C. elegans microbiota and potential pathogens can have widely divergent effects on Orsay virus transmission, such that associated bacteria can effectively determine host susceptibility versus resistance to viral infection. Our study provides quantitative evidence for a critical role for tripartite host-virus-bacteria interactions in determining the transmissibility of viruses among susceptible hosts.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Carlo Giannangelo, Matthew P Challis ... Darren J Creek
    Research Article

    New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.