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Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains

View ORCID ProfileTatsuaki Kurata, View ORCID ProfileChayan Kumar Saha, View ORCID ProfileJessica A. Buttress, View ORCID ProfileToomas Mets, View ORCID ProfileTetiana Brodiazhenko, View ORCID ProfileKathryn J. Turnbull, View ORCID ProfileOlolade F. Awoyomi, View ORCID ProfileSofia Raquel Alves Oliveira, View ORCID ProfileSteffi Jimmy, View ORCID ProfileKarin Ernits, View ORCID ProfileMaxence Delannoy, View ORCID ProfileKarina Persson, View ORCID ProfileTanel Tenson, View ORCID ProfileHenrik Strahl, View ORCID ProfileVasili Hauryliuk, View ORCID ProfileGemma C. Atkinson
doi: https://doi.org/10.1101/2021.05.07.442387
Tatsuaki Kurata
1Department of Experimental Medicine, University of Lund, 221 84 Lund, Sweden
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Chayan Kumar Saha
2Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden
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Jessica A. Buttress
3Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, United Kingdom
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Toomas Mets
4University of Tartu, Institute of Technology, 50411 Tartu, Estonia
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Tetiana Brodiazhenko
4University of Tartu, Institute of Technology, 50411 Tartu, Estonia
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Kathryn J. Turnbull
5Department of Clinical Microbiology, Rigshospitalet, 2200 Copenhagen, Denmark
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Ololade F. Awoyomi
6Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden
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Sofia Raquel Alves Oliveira
4University of Tartu, Institute of Technology, 50411 Tartu, Estonia
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Steffi Jimmy
7Centre for Structural Systems Biology (CSSB), Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany
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Karin Ernits
8Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
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Maxence Delannoy
9Département Génie Biologique, Campus SophiaTech, Université Nice Sophia Antipolis, France
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Karina Persson
8Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
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Tanel Tenson
4University of Tartu, Institute of Technology, 50411 Tartu, Estonia
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Henrik Strahl
3Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, United Kingdom
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Vasili Hauryliuk
1Department of Experimental Medicine, University of Lund, 221 84 Lund, Sweden
2Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden
4University of Tartu, Institute of Technology, 50411 Tartu, Estonia
10Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, 901 87 Umeå, Sweden
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  • For correspondence: gemma.atkinson@med.lu.se vasili.hauryliuk@med.lu.se
Gemma C. Atkinson
1Department of Experimental Medicine, University of Lund, 221 84 Lund, Sweden
2Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden
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  • For correspondence: gemma.atkinson@med.lu.se vasili.hauryliuk@med.lu.se
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Abstract

Toxin-Antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids, as well as bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets, and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analysed the conservation of genomic neighbourhoods using our computational tool FlaGs (for Flanking Genes), which allows high-throughput detection of TA-like operons. Focussing on the widespread but poorly experimentally characterised antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have renamed the domain to Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralised TA pairs. While the majority of validated PanA-neutralised toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises replication and translation, as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralisation mechanisms employed by Type II TA systems. Finally, through directed evolution we have selected PanA variants that can neutralise non-cognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

Significance Toxin-antitoxin systems are enigmatic and diverse elements of bacterial and bacteriophage genomes. We have uncovered remarkable versatility of an antitoxin protein domain, that has evolved to neutralise dozens of different toxin domains. We find that antitoxins carrying this domain – Panacea – form complexes with their cognate toxins, indicating a direct neutralisation mechanism, and that Panacea can be evolved to neutralise a non-cognate and non-homologous toxin with just two amino acid substitutions. This raises the possibility that this domain could be an adaptable universal, or semi-universal protein neutraliser with significant biotechnological and medical potential.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains
Tatsuaki Kurata, Chayan Kumar Saha, Jessica A. Buttress, Toomas Mets, Tetiana Brodiazhenko, Kathryn J. Turnbull, Ololade F. Awoyomi, Sofia Raquel Alves Oliveira, Steffi Jimmy, Karin Ernits, Maxence Delannoy, Karina Persson, Tanel Tenson, Henrik Strahl, Vasili Hauryliuk, Gemma C. Atkinson
bioRxiv 2021.05.07.442387; doi: https://doi.org/10.1101/2021.05.07.442387
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Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains
Tatsuaki Kurata, Chayan Kumar Saha, Jessica A. Buttress, Toomas Mets, Tetiana Brodiazhenko, Kathryn J. Turnbull, Ololade F. Awoyomi, Sofia Raquel Alves Oliveira, Steffi Jimmy, Karin Ernits, Maxence Delannoy, Karina Persson, Tanel Tenson, Henrik Strahl, Vasili Hauryliuk, Gemma C. Atkinson
bioRxiv 2021.05.07.442387; doi: https://doi.org/10.1101/2021.05.07.442387

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