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J. Biol. Chem., Vol. 280, Issue 40, 34245-34258, October 7, 2005

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Proteomic Analysis of Rhoptry Organelles Reveals Many Novel Constituents for Host-Parasite Interactions in Toxoplasma gondii^*

Peter J. Bradley, Chris Ward¶, Stephen J. Cheng, David L. Alexander, Susan Coller, Graham H. Coombs¶, Joe Dan Dunn, David J. Ferguson||, Sanya J. Sanderson**, Jonathan M. Wastling¶**1, and John C. Boothroyd12

From the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, the Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California 90095, the ^¶Division of Infection and Immunity, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, the ^**Departments of Pre-clinical Veterinary Science and Veterinary Pathology, University of Liverpool, Liverpool L69 7ZJ, United Kingdom, and ^||Nuffield Department of Pathology, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.

Received for publication, April 18, 2004 , and in revised form, July 5, 2005.

^* This work was supported by National Institutes of Health Grants RO1AI 21423, RO1AI45057 (to J. C. B.), F32AI10552 (to D. L. A.), T32GM07276 (to S. C.), and T32GM07276 (to J. D. D.), Howard Hughes Medical Institute predoctoral award (to J. D. D.), American Cancer Society Grant PF-99-018-01-MBC, Ellison Medical Foundation Grant ID-NS-0162-04 (to P. J. B.), and the Biotechnology and Biological Sciences Research Council Grants 17/S13819 and BBS/B/03807 (to J. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 650-723-7984; Fax: 650-723-6853; E-mail: john.boothroyd{at}stanford.edu.

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