Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration

  1. Malgorzata Broncel
  2. Caia Dominicus
  3. Luis Vigetti
  4. Stephanie D Nofal
  5. Edward J Bartlett
  6. Bastien Touquet
  7. Alex Hunt
  8. Bethan Alexandra Wallbank
  9. Stefania Federico
  10. Stephen Matthews
  11. Joanna Claire Young
  12. Edward W Tate
  13. Isabelle Tardieux
  14. Moritz Treeck  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom
  2. Université Grenoble Alpes, France
  3. Imperial College London, United Kingdom

Abstract

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 5, 6 and 7. Source data for mass spectrometry proteomics results can be found in Supplementary files 1-4. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD019677.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Malgorzata Broncel

    Signalling in Apicomplexan Parasites Lab, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2991-3500
  2. Caia Dominicus

    Signalling in Apicomplexan Parasites Lab, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
  3. Luis Vigetti

    Institute for Advanced Biosciences, Université Grenoble Alpes, Grenoble, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9733-2770
  4. Stephanie D Nofal

    Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1415-3369
  5. Edward J Bartlett

    Department of Chemistry, Imperial College London, London, United Kingdom
    Competing interests
    No competing interests declared.
  6. Bastien Touquet

    Team Membrane and Cell Dynamics of Host Parasite Interactions, Université Grenoble Alpes, Grenoble, France
    Competing interests
    No competing interests declared.
  7. Alex Hunt

    Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7431-7156
  8. Bethan Alexandra Wallbank

    Signalling in Apicomplexan Parasites Lab, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6432-2135
  9. Stefania Federico

    Peptide Synthesis STP, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
  10. Stephen Matthews

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0676-0927
  11. Joanna Claire Young

    Signalling in Apicomplexan Parasites Lab, The Francis Crick Institute, London, United Kingdom
    Competing interests
    No competing interests declared.
  12. Edward W Tate

    Department of Chemistry, Imperial College London, London, United Kingdom
    Competing interests
    Edward W Tate, EWT is a founder, shareholder and Director of Myricx Pharma Ltd.
  13. Isabelle Tardieux

    Team Membrane and Cell Dynamics of Host Parasite Interactions, Université Grenoble Alpes, Grenoble, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5677-7463
  14. Moritz Treeck

    Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, London, United Kingdom
    For correspondence
    moritz.treeck@crick.ac.uk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9727-6657

Funding

Francis Crick Institute (FC001189)

  • Malgorzata Broncel
  • Caia Dominicus
  • Stephanie D Nofal
  • Alex Hunt
  • Bethan Alexandra Wallbank
  • Joanna Claire Young
  • Moritz Treeck

NIH Office of the Director (R01AI123457)

  • Malgorzata Broncel
  • Caia Dominicus
  • Moritz Treeck

Leverhulme Trust (RPG-2018-107)

  • Stephen Matthews

Cancer Research UK (C29637/A20183)

  • Edward J Bartlett
  • Edward W Tate

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

Reviewing Editor

  1. Dominique Soldati-Favre, University of Geneva, Switzerland

Publication history

  1. Received: April 14, 2020
  2. Accepted: June 27, 2020
  3. Accepted Manuscript published: July 3, 2020 (version 1)
  4. Version of Record published: July 21, 2020 (version 2)

Copyright

© 2020, Broncel 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

  • 1,756
    Page views
  • 271
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Malgorzata Broncel
  2. Caia Dominicus
  3. Luis Vigetti
  4. Stephanie D Nofal
  5. Edward J Bartlett
  6. Bastien Touquet
  7. Alex Hunt
  8. Bethan Alexandra Wallbank
  9. Stefania Federico
  10. Stephen Matthews
  11. Joanna Claire Young
  12. Edward W Tate
  13. Isabelle Tardieux
  14. Moritz Treeck
(2020)
Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration
eLife 9:e57861.
https://doi.org/10.7554/eLife.57861

Further reading

    1. Computational and Systems Biology
    2. Microbiology and Infectious Disease
    Chiara Vanni et al.
    Research Article Updated

    Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40–60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we present a conceptual framework, its translation into the computational workflow AGNOSTOS and a demonstration on how we can bridge the known-unknown gap in genomes and metagenomes. By analyzing 415,971,742 genes predicted from 1749 metagenomes and 28,941 bacterial and archaeal genomes, we quantify the extent of the unknown fraction, its diversity, and its relevance across multiple organisms and environments. The unknown sequence space is exceptionally diverse, phylogenetically more conserved than the known fraction and predominantly taxonomically restricted at the species level. From the 71 M genes identified to be of unknown function, we compiled a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria (also known as Candidate Phyla Radiation, CPR), which provides a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Florian Bleffert et al.
    Research Article Updated

    Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.