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Published March 1, 2022 | Submitted + Supplemental Material
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An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

Abstract

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. In contrast to previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associated with the exocytic site - prior to invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarity to GPCR proteins. We propose that the CRMP complex acts as host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We thank Sebastian Lourido for the pU6-Universal plasmid, Dominique Soldati-Favre for providing the anti-ARM (ARO) antibodies and pLinker-2xTy-DHFR plasmid, Nicolas Dos Santos Pacheco for helping in setting up the Ultrastructure Expansion Microscopy, Anita Koshy for the toxofilin-Cre plasmid and Helen Blau's lab for the Cre reporter DSred cell line. We thank Veronique Richard and Frank Godiard of the MEA platform, University of Montpellier for their assistance with electron microscopy and Pilar Ruga Fahy of the Pôle Facultaire de Microscopie Ultrastructurale, in Geneva for preparation of freeze-fracture replicas. We are also grateful to Elodie Jublanc, Vicky Diakou and the imaging facility MRI at the University of Montpellier, part of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»), and Christophe Duperray of the MRI-Cytometry at the Institute for Regenerative Medicine and Biotherapy for their assistance and technical support. Mass spectrometry experiments were carried out using the facilities of the Montpellier Proteomics Platform (PPM, BioCampus Montpellier). We thank Stefan Steimle for his technical assistance with the Krios G3i cryogenic electron microscope; the Singh Center for Nanotechnology and the Beckman Center for Cryogenic Electron Microscopy at the University of Pennsylvania for hosting and supporting the use of the Titan Krios. Dr Maryse Lebrun is an INSERM researcher. This work was supported by the Laboratoire d'Excellence (LabEx) (ParaFrap ANR-11-LABX-0024), and European Research Council (ERC advanced grant number 833309 KissAndSpitRhoptry) to M.L.; by the FACCTS (France and Chicago Collaborating in the Sciences) to A.P.T and M.L. ; by NIH GM105783 to A.P.T. ; by a David and Lucile Packard Fellowship for Science and Engineering (2019-69645) and a Pennsylvania Department of Health FY19 Health Research Formula Fund to Y.-W.C; by NIH R01 AI112427 to B. S. D.S. and M.M.C. are supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under Grant agreement no. 833309 to M.L. AUTHOR CONTRIBUTION. Conceptualization: D.S. M.L. Investigation Tetrahymena: L.M.T. performed CDH analysis; D.S. generated and analyzed mutants. Investigation Toxoplasma: J.D., J.H., D.M.P.V. generated the tagged and iKD lines; J.D. performed phenotypic analysis of the mutants with the help of M.M., D.M.P.V., J.H.; D.S. generated and analyzed lines for co-IP and fluorescence microscopy of extracellular parasites; D.M.P.V., S.U., performed IP and mass spectrometry analysis; L.B.S., J.H. performed EM analysis; D.S., M.M.C., performed ultrastructure expansion microscopy. Phylogenies: D.S. Freeze fracture data: D.S., J.H. prepared samples; D.S., J.F.D., collected data. Cryo-ET data: A.G. cultured the cells; A.G. and S.K.M. prepared the grids; S.K.M. collected data; S.K.M. and L.T. performed analysis on the tomograms and prepared figures with guidance from Y.-W.C.; L.T. performed subtomogram averaging for the CRMPb-iKD mutant with input from Y.-W.C. and S.K.M. Writing-Original draft: D.S., M.L. Visualization: D.S., J.D., M.L. Writing-Review & Editing: A.P.T., B.S., J.D., S.K.M, with inputs from all authors. Supervision: A.P.T., Y.-W.C., M.L. Funding: B.S., A.P.T., Y.-W.C., M.L. DATA AVAILABILITY. 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 PXD031161 and PXD031164. The authors have declared no competing interest.

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Submitted - 2022.02.25.481937v1.full.pdf

Supplemental Material - media-1.pdf

Supplemental Material - media-2.pdf

Supplemental Material - media-3.pdf

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Additional details

Created:
August 20, 2023
Modified:
October 23, 2023