An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

  1. Michael J Coffey
  2. Brad E Sleebs
  3. Alessandro D Uboldi
  4. Alexandra L Garnham
  5. Magdalena Franco
  6. Nicole D Marino
  7. Michael W Panas
  8. David JP Ferguson
  9. Marta Enciso
  10. Matthew T O'Neill
  11. Sash Lopaticki
  12. Rebecca J Stewart
  13. Grant Dewson
  14. Gordon K Smyth
  15. Brian J Smith
  16. Seth L Masters
  17. John C Boothroyd
  18. Justin A Boddey
  19. Christopher J Tonkin  Is a corresponding author
  1. The Walter and Eliza Hall Institute of Medical Research, Australia
  2. Stanford University School of Medicine, United States
  3. University of Oxford, United Kingdom
  4. La Trobe University, Australia
  5. Walter and Eliza Hall Institute of Medical Research, Australia

Abstract

Infection by Toxoplasma gondii leads to massive changes to the host cell. Here we identify a novel host cell effector export pathway, which requires the Golgi-resident Aspartyl Protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

Article and author information

Author details

  1. Michael J Coffey

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  2. Brad E Sleebs

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Alessandro D Uboldi

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Alexandra L Garnham

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Magdalena Franco

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Nicole D Marino

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Michael W Panas

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. David JP Ferguson

    Nuffield Department of Clinical Laboratory Science, University of Oxford, Oxoford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Marta Enciso

    La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  10. Matthew T O'Neill

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  11. Sash Lopaticki

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  12. Rebecca J Stewart

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  13. Grant Dewson

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  14. Gordon K Smyth

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  15. Brian J Smith

    La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  16. Seth L Masters

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  17. John C Boothroyd

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Justin A Boddey

    The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  19. Christopher J Tonkin

    Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
    For correspondence
    tonkin@wehi.edu.au
    Competing interests
    The authors declare that no competing interests exist.

Ethics

Animal experimentation: All animal experiments complied with the regulatory standards of and were approved by the Walter and Eliza Hall Institute Animal Ethics Committees under approval number 2014.019.

Copyright

© 2015, Coffey 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

  • 4,233
    views
  • 902
    downloads
  • 98
    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. Michael J Coffey
  2. Brad E Sleebs
  3. Alessandro D Uboldi
  4. Alexandra L Garnham
  5. Magdalena Franco
  6. Nicole D Marino
  7. Michael W Panas
  8. David JP Ferguson
  9. Marta Enciso
  10. Matthew T O'Neill
  11. Sash Lopaticki
  12. Rebecca J Stewart
  13. Grant Dewson
  14. Gordon K Smyth
  15. Brian J Smith
  16. Seth L Masters
  17. John C Boothroyd
  18. Justin A Boddey
  19. Christopher J Tonkin
(2015)
An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell
eLife 4:e10809.
https://doi.org/10.7554/eLife.10809

Share this article

https://doi.org/10.7554/eLife.10809

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

    1. Microbiology and Infectious Disease
    Yue Sun, Jingwei Li ... Xin Deng
    Research Article

    The model Gram-negative plant pathogen Pseudomonas syringae utilises hundreds of transcription factors (TFs) to regulate its functional processes, including virulence and metabolic pathways that control its ability to infect host plants. Although the molecular mechanisms of regulators have been studied for decades, a comprehensive understanding of genome-wide TFs in Psph 1448A remains limited. Here, we investigated the binding characteristics of 170 of 301 annotated TFs through chromatin immunoprecipitation sequencing (ChIP-seq). Fifty-four TFs, 62 TFs, and 147 TFs were identified in top-level, middle-level, and bottom-level, reflecting multiple higher-order network structures and direction of information flow. More than 40,000 TF pairs were classified into 13 three-node submodules which revealed the regulatory diversity of TFs in Psph 1448A regulatory network. We found that bottom-level TFs performed high co-associated scores to their target genes. Functional categories of TFs at three levels encompassed various regulatory pathways. Three and 25 master TFs were identified to involve in virulence and metabolic regulation, respectively. Evolutionary analysis and topological modularity network revealed functional variability and various conservation of TFs in P. syringae (Psph 1448A, Pst DC3000, Pss B728a, and Psa C48). Overall, our findings demonstrated a global transcriptional regulatory network of genome-wide TFs in Psph 1448A. This knowledge can advance the development of effective treatment and prevention strategies for related infectious diseases.