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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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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Scientific Reports (2017)