A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite

  1. Abhinay Ramaprasad
  2. Paul-Christian Burda
  3. Enrica Calvani
  4. Aaron J Sait
  5. Susana Alejandra Palma-Duran
  6. Chrislaine Withers-Martinez
  7. Fiona Hackett
  8. James Macrae
  9. Lucy Collinson
  10. Tim Wolf Gilberger  Is a corresponding author
  11. Michael J Blackman  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom
  2. Centre for Structural Systems Biology, Germany
  3. Bernhard Nocht Institute for Tropical Medicine, Germany

Abstract

The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.

Data availability

Sequencing data have been deposited in ENA under Project PRJEB55180.All data generated or analysed are included in the manuscript or provided as source data files.All source codes are available via github - https://github.com/a2g1n/GDPDxcute.

The following data sets were generated

Article and author information

Author details

  1. Abhinay Ramaprasad

    Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9372-5526
  2. Paul-Christian Burda

    Centre for Structural Systems Biology, Hamburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0461-4352
  3. Enrica Calvani

    Mass Spectrometry Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Aaron J Sait

    Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6091-0426
  5. Susana Alejandra Palma-Duran

    Mass Spectrometry Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Chrislaine Withers-Martinez

    Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Fiona Hackett

    Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. James Macrae

    Mass Spectrometry Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1464-8583
  9. Lucy Collinson

    Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Tim Wolf Gilberger

    Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
    For correspondence
    gilberger@bnitm.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7965-8272
  11. Michael J Blackman

    Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom
    For correspondence
    Mike.Blackman@crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7442-3810

Funding

H2020 Marie Skłodowska-Curie Actions (751865)

  • Abhinay Ramaprasad

Wellcome Trust (20318/A/20/Z)

  • Michael J Blackman

Cancer Research UK (CC2129)

  • Abhinay Ramaprasad
  • Enrica Calvani
  • Aaron J Sait
  • Susana Alejandra Palma-Duran
  • Chrislaine Withers-Martinez
  • Fiona Hackett
  • James Macrae
  • Lucy Collinson
  • Michael J Blackman

Medical Research Council (CC2129)

  • Abhinay Ramaprasad
  • Enrica Calvani
  • Aaron J Sait
  • Susana Alejandra Palma-Duran
  • Chrislaine Withers-Martinez
  • Fiona Hackett
  • James Macrae
  • Lucy Collinson
  • Michael J Blackman

Wellcome Trust (CC2129)

  • Abhinay Ramaprasad
  • Enrica Calvani
  • Aaron J Sait
  • Susana Alejandra Palma-Duran
  • Chrislaine Withers-Martinez
  • Fiona Hackett
  • James Macrae
  • Lucy Collinson
  • Michael J Blackman

Wellcome Trust (ISSF2)

  • Michael J Blackman

Deutsche Forschungsgemeinschaft (414222880)

  • Paul-Christian Burda

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

Reviewing Editor

  1. Malcolm J McConville, The University of Melbourne, Australia

Version history

  1. Preprint posted: June 14, 2022 (view preprint)
  2. Received: July 27, 2022
  3. Accepted: December 23, 2022
  4. Accepted Manuscript published: December 28, 2022 (version 1)
  5. Version of Record published: January 30, 2023 (version 2)

Copyright

© 2022, Ramaprasad 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

  • 932
    views
  • 191
    downloads
  • 9
    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. Abhinay Ramaprasad
  2. Paul-Christian Burda
  3. Enrica Calvani
  4. Aaron J Sait
  5. Susana Alejandra Palma-Duran
  6. Chrislaine Withers-Martinez
  7. Fiona Hackett
  8. James Macrae
  9. Lucy Collinson
  10. Tim Wolf Gilberger
  11. Michael J Blackman
(2022)
A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite
eLife 11:e82207.
https://doi.org/10.7554/eLife.82207

Share this article

https://doi.org/10.7554/eLife.82207

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Thomas RM Germe, Natassja G Bush ... Anthony Maxwell
    Research Article

    DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).