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Efficient invasion by Toxoplasma depends on the subversion of host protein networks

Nature Microbiology volume 2, pages 1358–1366 (2017)Cite this article

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Abstract

Apicomplexan parasites are important pathogens of humans and domestic animals, including Plasmodium species (the agents of malaria) and Toxoplasma gondii, which is responsible for toxoplasmosis. They replicate within the cells of their animal hosts, to which they gain access using a unique parasite-driven invasion process. At the core of the invasion machine is a structure at the interface between the invading parasite and host cell called the moving junction (MJ)1. The MJ serves as both a molecular doorway to the host cell and an anchor point enabling the parasite to engage its motility machinery to drive the penetration of the host cell2, ultimately yielding a protective vacuole3. The MJ is established through self-assembly of parasite proteins at the parasite–host interface4. However, it is unknown whether host proteins are subverted for MJ formation. Here, we show that Toxoplasma parasite rhoptry neck proteins (RON2, RON4 and RON5) cooperate to actively recruit the host CIN85, CD2AP and the ESCRT-I components ALIX and TSG101 to the MJ during invasion. We map the interactions in detail and demonstrate that the parasite mimics and subverts conserved binding interfaces with remarkable specificity. Parasite mutants unable to recruit these host proteins show inefficient host cell invasion in culture and attenuated virulence in mice. This study reveals molecular mechanisms by which parasites subvert widely conserved host machinery to force highly efficient host cell access.

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Fig. 1: RON4 recruits ALIX and CIN85 at the MJ.
Fig. 2: RON4 and RON5 play a role in invasion by binding ALIX, CIN85/CD2AP and TSG101.
Fig. 3: ALIX and TSG101 play a role in virulence.
Fig. 4: Model of the host cell part of the MJ.

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References

  1. Aikawa, M., Miller, L. H., Johnson, J. & Rabbege, J. Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite. J. Cell. Biol. 77, 72–82 (1978).

    Article  CAS  PubMed  Google Scholar 

  2. Bichet, M. et al. The Toxoplasma–host cell junction is anchored to the cell cortex to sustain parasite invasive force. BMC Biol. 12, 773 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Mordue, D. G., Desai, N., Dustin, M. & Sibley, L. D. Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J. Exp. Med. 190, 1783–1792 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Besteiro, S., Dubremetz, J. F. & Lebrun, M. The moving junction of apicomplexan parasites: a key structure for invasion. Cell. Microbiol. 13, 797–805 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Alexander, D. L., Mital, J., Ward, G. E., Bradley, P. & Boothroyd, J. C. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog. 1, e17 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J. F. & Lebrun, M. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, e1000309 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lamarque, M. et al. The RON2–AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS Pathog. 7, e1001276 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tyler, J. S. & Boothroyd, J. C. The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex. PLoS Pathog. 7, e1001282 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tonkin, M. L. et al. Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science 333, 463–467 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Vulliez-Le Normand, B. et al. Structural and functional insights into the malaria parasite moving junction complex. PLoS Pathog. 8, e1002755 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Beck, J. R., Chen, A. L., Kim, E. W. & Bradley, P. J. RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS Pathog. 10, e1004025 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Lamarque, M. H. et al. Plasticity and redundancy among AMA–RON pairs ensure host cell entry of Toxoplasma parasites. Nat. Commun. 5, 4098 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Bissig, C. & Gruenberg, J. ALIX and the multivesicular endosome: ALIX in Wonderland. Trends Cell. Biol. 24, 19–25 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Dikic, I. CIN85/CMS family of adaptor molecules. FEBS Lett. 529, 110–115 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Fisher, R. D. et al. Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding. Cell 128, 841–852 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Kowanetz, K. et al. Identification of a novel proline–arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors. J. Biol. Chem. 278, 39735–39746 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Dustin, M. L. et al. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667–677 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Hurley, J. H. ESCRTs are everywhere. EMBO J. 34, 2398–2407 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Garrus, J. E. et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107, 55–65 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Segura-Morales, C. et al. Tsg101 and Alix interact with murine leukemia virus Gag and cooperate with Nedd4 ubiquitin ligases during budding. J. Biol. Chem. 280, 27004–27012 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Urata, S., Yokosawa, H. & Yasuda, J. Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix. Virol. J. 4, 66 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Mordue, D. G. & Sibley, L. D. A novel population of Gr-1+-activated macrophages induced during acute toxoplasmosis. J. Leukoc. Biol. 74, 1015–1025 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Schmidt, M. H., Chen, B., Randazzo, L. M. & Bogler, O. SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion. J. Cell. Sci. 116, 2845–2855 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Campos, Y. et al. Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier. Nat. Commun. 7, 11876 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Johnson, R. I., Seppa, M. J. & Cagan, R. L. The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. J. Cell. Biol. 180, 1191–1204 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gonzalez, V. et al. Host cell entry by Apicomplexa parasites requires actin polymerization in the host cell. Cell Host Microbe 5, 259–272 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Sweeney, K. R., Morrissette, N. S., LaChapelle, S. & Blader, I. J. Host cell invasion by Toxoplasma gondii is temporally regulated by the host microtubule cytoskeleton. Eukaryot. Cell 9, 1680–1689 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tang, V. W. & Brieher, W. M. FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions. J. Cell. Biol. 203, 815–833 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Veiga, E. & Cossart, P. Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells. Nat. Cell Biol. 7, 894–900 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Pan, S. et al. Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly. J. Biol. Chem. 281, 34640–34650 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Huynh, M. H. & Carruthers, V. B. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot. Cell 8, 530–539 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sheiner, L. et al. A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor. PLoS Pathog. 7, e1002392 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sidik, S. M., Hackett, C. G., Tran, F., Westwood, N. J. & Lourido, S. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS ONE 9, e100450 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Im, Y. J. et al. Crystallographic and functional analysis of the ESCRT-I/HIV-1 Gag PTAP interaction. Structure 18, 1536–1547 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Vojtek, A. B. & Hollenberg, S. M. Ras–Raf interaction: two-hybrid analysis. Methods Enzymol. 255, 331–342 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Fromont-Racine, M., Rain, J. C. & Legrain, P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat. Genet. 16, 277–282 (1997).

    Article  CAS  PubMed  Google Scholar 

  37. Formstecher, E. et al. Protein interaction mapping: a Drosophila case study. Genome Res. 15, 376–384 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wojcik, J., Boneca, I. G. & Legrain, P. Prediction, assessment and validation of protein interaction maps in bacteria. J. Mol. Biol. 323, 763–770 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Rain, J. C. et al. The protein–protein interaction map of Helicobacter pylori. Nature 409, 211–215 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. London, N., Raveh, B., Cohen, E., Fathi, G. & Schueler-Furman, O. Rosetta FlexPepDock web server—high resolution modeling of peptide–protein interactions. Nucleic Acids Res. 39, W249–W253 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Raveh, B., London, N. & Schueler-Furman, O. Sub-angstrom modeling of complexes between flexible peptides and globular proteins. Proteins 78, 2029–2040 (2010).

    CAS  PubMed  Google Scholar 

  42. Crawford, J., Tonkin, M. L., Grujic, O. & Boulanger, M. J. Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii. J. Biol. Chem. 285, 15644–15652 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lebrun, M. et al. The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion. Cell. Microbiol. 7, 1823–1833 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Lamarque, M. H. et al. Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii. PLoS ONE 7, e32457 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank P. Soubeyran for the pcDNA3-Flag-CIN85 FL plasmid, W. Sundquist for the GFP–ALIX, GST–ALIX, CHMP4B–GFP, GFP–VPS4, myc-VPS28, myc-VPS37 and myc-CD2AP plasmids, B. Striepen and M.J. Cipriano for the pU6-Cas9-YFP plasmid, P. Bradley for the anti-RON5C and S. Lourido for the pU6-universal plasmid. We are grateful to T. Soldati and B. Striepen for critical reading of the manuscript. We thank the staff of the MRI-Cytometry at the Institute for Regenerative Medicine and Biotherapy and the Flow Cytometry Facility at the Centre Médical Universitaire, Geneva, for sorting the parasites. This research was supported by the Agence Nationale de la Recherche (ANRA-12-BSV3-0012-01 and ANR-16-CE15-0010-01), Laboratoire d’Excellence (ParaFrap ANR-11-LABX-0024) and Fondation pour la Recherche Médicale (Equipe FRM DEQ20130326508) to M.L., the Canadian Institutes of Health Research grants 82915 and 148596 to M.J.B. and the Swiss National Science Foundation (FN3100A0-116722) to D.S.-F. A.G. was funded by the French Parasitology consortium ParaFrap (ANR-11-LABX-0024) and was a recipient of FRM (award no. FDT20160435387). D.J. is supported by Carigest SA. M.J.B. gratefully acknowledges the Canada Research Chairs program for salary support.

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Authors and Affiliations

  1. Unité Mixte de Recherche 5235, Centre National de la Recherche Scientifique, Université de Montpellier 2, 34095, Montpellier, France

    Amandine Guérin, Rosa Milagros Corrales, Mauld H. Lamarque & Maryse Lebrun

  2. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8P 5C2, Canada

    Michele L. Parker & Martin J. Boulanger

  3. Department of Microbiology and Molecular Medicine, Centre Médical Universitaire, University of Geneva, 1 Rue Michel-Servet, 1206, Geneva, Switzerland

    Damien Jacot & Dominique Soldati-Favre

  4. Department of Internal Medicine and Experimental Pathology, Immunology and Microbiology, American University of Beirut, Beirut, 1107 2020, Lebanon

    Hiba El Hajj

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Contributions

M.L. conceived the study. A.G. designed, performed and interpreted most of the experimental work. R.M.C., M.H.L., H.E.H. and M.L.P. designed and performed specific experimental work. D.J. and D.S.-F. provided technical support for the cell sorting of parasites. M.L. and M.J.B. supervised the research. A.G. and M.L. wrote the paper with editorial support from M.J.B., M.H.L., D.S.-F. and D.J. All authors commented on the manuscript.

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Correspondence to Maryse Lebrun.

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Supplementary Figures 1–11, full blot results, Supplementary Tables 1–3, Supplementary Text, Supplementary References.

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Guérin, A., Corrales, R.M., Parker, M.L. et al. Efficient invasion by Toxoplasma depends on the subversion of host protein networks. Nat Microbiol 2, 1358–1366 (2017). https://doi.org/10.1038/s41564-017-0018-1

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