Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context
- University of California, San Francisco, United States
- University of Miami, United States
- InBios International, United States
- Pacific Northwest University of Health Sciences, United States
- Stanford University, United States
- Washington University in St. Louis, United States
- Newcastle University, United Kingdom
- University of Washington, United States
- Carnegie Mellon University, United States
- University of California, San Diego, United States
- Université Grenoble Alpes, France
- 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
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T6S amidase effector 1 deep mutational scanningNCBI, Sequence Read Archive, PRJNA803461.
Article and author information
Author details
William L Hatleberg
Jean-Pierre Simorre
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
- Petra Anne Levin, Washington University in St. Louis, United States
Version history
- Preprint posted: February 16, 2022 (view preprint)
- Received: May 22, 2022
- Accepted: June 23, 2022
- Accepted Manuscript published: June 28, 2022 (version 1)
- 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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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
