Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

  1. Atanas Radkov
  2. Anne L Sapiro
  3. Sebastian Flores
  4. Corey Henderson
  5. Hayden Saunders
  6. Rachel Kim
  7. Steven Massa
  8. Samuel Thompson
  9. Chase Mateusiak
  10. Jacob Biboy
  11. Ziyi Zhao
  12. Lea M Starita
  13. William L Hatleberg
  14. Waldemar Vollmer
  15. Alistair B Russell
  16. Jean-Pierre Simorre
  17. Spencer Anthony-Cahill
  18. Peter Brzovic
  19. Beth Hayes
  20. Seemay Chou  Is a corresponding author
  1. University of California, San Francisco, United States
  2. University of Miami, United States
  3. InBios International, United States
  4. Pacific Northwest University of Health Sciences, United States
  5. Stanford University, United States
  6. Washington University in St. Louis, United States
  7. Newcastle University, United Kingdom
  8. University of Washington, United States
  9. Carnegie Mellon University, United States
  10. University of California, San Diego, United States
  11. Université Grenoble Alpes, France
  12. Western Washington University, United States

Abstract

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

Data availability

-Deep mutational scanning dataset has been deposited to NCBI, Sequence Read Archive - identifier: PRJNA803461-X-ray structure crystallography dataset has been deposited to PDB - ID: 7TVH-NMR resonance peak assignments have been directly provided in the manuscript-Custom scripts for DMS analyses have been uploaded at GitHub at https://github.com/AtanasDRadkov/ChouLab_DMS-All other data generated or analyzed in this study are included in the manuscript and supporting files

The following data sets were generated

Article and author information

Author details

  1. Atanas Radkov

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  2. Anne L Sapiro

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6612-8272
  3. Sebastian Flores

    University of Miami, Miami, United States
    Competing interests
    No competing interests declared.
  4. Corey Henderson

    InBios International, Seattle, United States
    Competing interests
    No competing interests declared.
  5. Hayden Saunders

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7582-3031
  6. Rachel Kim

    Pacific Northwest University of Health Sciences, Yakima, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3793-4264
  7. Steven Massa

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  8. Samuel Thompson

    Department of Bioengineering, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  9. Chase Mateusiak

    Computer Science Department, Washington University in St. Louis, St. Louis, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2890-4242
  10. Jacob Biboy

    Centre for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1286-6851
  11. Ziyi Zhao

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  12. Lea M Starita

    Department of Genome Sciences, University of Washington, Seattle, United States
    Competing interests
    No competing interests declared.
  13. William L Hatleberg

    Carnegie Mellon University, Pittsburgh, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0423-7123
  14. Waldemar Vollmer

    Center for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, United Kingdom
    Competing interests
    No competing interests declared.
  15. Alistair B Russell

    Division of Biological Sciences, University of California, San Diego, La Jolla, United States
    Competing interests
    No competing interests declared.
  16. Jean-Pierre Simorre

    Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7943-1342
  17. Spencer Anthony-Cahill

    Chemistry Department, Western Washington University, Seattle, United States
    Competing interests
    No competing interests declared.
  18. Peter Brzovic

    Department of Biochemistry, University of Washington, Seattle, United States
    Competing interests
    No competing interests declared.
  19. Beth Hayes

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6633-751X
  20. Seemay Chou

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    seemay.chou@ucsf.edu
    Competing interests
    Seemay Chou, currently the President and CEO of Arcadia Science, a for-profit research company focused on non-model organisms. While work presented in this manuscript is not related and will not be continued at Arcadia, I thought it would be prudent to declare this position which may be perceived by some as a competing interest..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7271-303X

Funding

IR-RMN-THC FR3050 CNRS

  • Jean-Pierre Simorre

Chan Zuckerberg Biohub

  • Seemay Chou

Sanghvi-Agarwal Innovation Award

  • Seemay Chou

Life Sciences Research Foundation

  • Anne L Sapiro

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Petra Anne Levin, Washington University in St. Louis, United States

Publication history

  1. Preprint posted: February 16, 2022 (view preprint)
  2. Received: May 22, 2022
  3. Accepted: June 23, 2022
  4. Accepted Manuscript published: June 28, 2022 (version 1)
  5. Version of Record published: July 8, 2022 (version 2)

Copyright

© 2022, Radkov 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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  1. Atanas Radkov
  2. Anne L Sapiro
  3. Sebastian Flores
  4. Corey Henderson
  5. Hayden Saunders
  6. Rachel Kim
  7. Steven Massa
  8. Samuel Thompson
  9. Chase Mateusiak
  10. Jacob Biboy
  11. Ziyi Zhao
  12. Lea M Starita
  13. William L Hatleberg
  14. Waldemar Vollmer
  15. Alistair B Russell
  16. Jean-Pierre Simorre
  17. Spencer Anthony-Cahill
  18. Peter Brzovic
  19. Beth Hayes
  20. Seemay Chou
(2022)
Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context
eLife 11:e79796.
https://doi.org/10.7554/eLife.79796
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