Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

Abstract

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided that cover all figures and give raw data, averages, statistics etc.

Article and author information

Author details

  1. Ali Alghamdi

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Jane C Munday

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Gustavo Daniel Campagnaro

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6542-0485
  4. Dominik Gurvic

    2.Computational Biology Centre for Translational and Interdisciplinary Research, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Fredrik Svensson

    Chemoinformatics, IOTA Pharmaceuticals Ltd, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Chinyere E Okpara

    Department of Chemistry, University of Liverpool, Liverpool, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Arvind Kumar

    Chemistry Department, Georgia State University, Atalanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Juan Quintana

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Maria Esther Martin Abril

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Patrik Milić

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Laura Watson

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Daniel Paape

    The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation,, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. Luca Settimo

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Anna Dimitriou

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Joanna Wielinska

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  16. Graeme Smart

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  17. Laura F Anderson

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  18. Christopher M Woodley

    Department of Chemistry, University of Liverpool, Liverpool, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  19. Siu Pui Ying Kelly

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  20. Hasan MS Ibrahim

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  21. Fabian Hulpia

    Laboratory for Medicinal Chemistry, University of Ghent, Ghent, Belgium
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7470-3484
  22. Mohammed I Al-Salabi

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  23. Anthonius A Eze

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4821-1689
  24. Teresa Sprenger

    Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  25. Ibrahim A Teka

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  26. Simon Gudin

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  27. Simone Weyand

    Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  28. Mark C Field

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  29. Christophe Dardonville

    Instituto de Química Médica, CSIC, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  30. Richard R Tidwell

    Department of Pathology and Lab Medicine, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  31. Mark Carrington

    Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6435-7266
  32. Paul M O'Neill

    Department of Chemistry, University of Liverpool, Liverpool, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  33. David W Boykin

    Chemistry Department, Georgia State University, Atalanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  34. Ulrich Zachariae

    School of Life Sciences / School of Science and Engineering, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  35. Harry P De Koning

    Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
    For correspondence
    Harry.De-Koning@glasgow.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9963-1827

Funding

Medical Research Council (G0701258)

  • Harry P De Koning

National Institutes of Health (GM111749)

  • David W Boykin

Medical Research Council (MR/R015791/1)

  • Harry P De Koning

Scottish Universities Physics Alliance

  • Ulrich Zachariae

Albaha University, Saudi Arabia

  • Ali Alghamdi

Science Without Borders, Brazil (206385/2014-5)

  • Gustavo Daniel Campagnaro

Wellcome (204697/Z/16/Z)

  • Mark C Field

Medical Research Council

  • Teresa Sprenger

Wellcome Trust and Royal Society (Sir Henry Dale fellowship)

  • Simone Weyand

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

Reviewing Editor

  1. Christine Clayton, DKFZ-ZMBH Alliance, Germany

Version history

  1. Received: February 26, 2020
  2. Accepted: August 6, 2020
  3. Accepted Manuscript published: August 7, 2020 (version 1)
  4. Accepted Manuscript updated: August 11, 2020 (version 2)
  5. Version of Record published: September 4, 2020 (version 3)

Copyright

© 2020, Alghamdi 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. Ali Alghamdi
  2. Jane C Munday
  3. Gustavo Daniel Campagnaro
  4. Dominik Gurvic
  5. Fredrik Svensson
  6. Chinyere E Okpara
  7. Arvind Kumar
  8. Juan Quintana
  9. Maria Esther Martin Abril
  10. Patrik Milić
  11. Laura Watson
  12. Daniel Paape
  13. Luca Settimo
  14. Anna Dimitriou
  15. Joanna Wielinska
  16. Graeme Smart
  17. Laura F Anderson
  18. Christopher M Woodley
  19. Siu Pui Ying Kelly
  20. Hasan MS Ibrahim
  21. Fabian Hulpia
  22. Mohammed I Al-Salabi
  23. Anthonius A Eze
  24. Teresa Sprenger
  25. Ibrahim A Teka
  26. Simon Gudin
  27. Simone Weyand
  28. Mark C Field
  29. Christophe Dardonville
  30. Richard R Tidwell
  31. Mark Carrington
  32. Paul M O'Neill
  33. David W Boykin
  34. Ulrich Zachariae
  35. Harry P De Koning
(2020)
Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei
eLife 9:e56416.
https://doi.org/10.7554/eLife.56416

Share this article

https://doi.org/10.7554/eLife.56416

Further reading

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    Chi-Ning Chuang, Hou-Cheng Liu ... Ting-Fang Wang
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    Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

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    2. Developmental Biology
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    Background:

    Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.

    Methods:

    Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry.

    Results:

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    This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.

    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.