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Jean-françois Dubremetz - One of the best experts on this subject based on the ideXlab platform.
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The Toxoplasma Protein ARO Mediates the Apical Positioning of Rhoptry Organelles, a Prerequisite for Host Cell Invasion
Cell host & microbe, 2013Co-Authors: Christina Mueller, Jean-françois Dubremetz, Natacha Klages, Damien Jacot, Joana M. Santos, Ana Cabrera, Tim W. Gilberger, Dominique Soldati-favreAbstract:Members of the phylum Apicomplexa actively enter host cells by a process involving the discharge of the apically localized microneme and Rhoptry organelles. To unravel the processes involved in Rhoptry organelle biogenesis, we focused on the Toxoplasma gondii armadillo repeats only protein (TgARO), a conserved acylated protein homogenously anchored to the Rhoptry membrane. Conditional disruption of TgARO results in the random cytosolic dispersion of rhoptries and a severe defect in T. gondii invasion, with no effects on intracellular growth or host cell egress. Importantly, Rhoptry displacement upon ARO depletion can be functionally complemented with wild-type TgARO but not an acylation mutant. TgARO interacts with myosin F, and inhibition of actin polymerization or myosin function also results in Rhoptry dispersal, indicating that the apical positioning of rhoptries is an actomyosin-based process. Thus, TgARO mediates the apical localization of rhoptries, which is specifically required for host cell invasion.
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Identification of a new Rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii.
PloS one, 2012Co-Authors: Mauld H. Lamarque, Jean-françois Dubremetz, Gaelle Lentini, Julien Papoin, Anne-laure Finizio, Alexander W. Pfaff, Ermanno Candolfi, Maryse LebrunAbstract:Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains Rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains Rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of Rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
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identification of a new Rhoptry neck complex ron9 ron10 in the apicomplexa parasite toxoplasma gondii
PLOS ONE, 2012Co-Authors: Mauld H. Lamarque, Jean-françois Dubremetz, Gaelle Lentini, Julien Papoin, Anne-laure Finizio, Alexander W. Pfaff, Ermanno Candolfi, Maryse LebrunAbstract:Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains Rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains Rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of Rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
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Molecular characterisation of a Cryptosporidium parvum Rhoptry protein candidate related to the Rhoptry neck proteins TgRON1 of Toxoplasma gondii and PfASP of Plasmodium falciparum.
Molecular and Biochemical Parasitology, 2012Co-Authors: Elisabetta Valentini, Simona Cherchi, Alessia Possenti, Jean-françois Dubremetz, Edoardo Pozio, Furio SpanoAbstract:Abstract Given the lack of knowledge on the Rhoptry proteins of Cryptosporidium parvum , we searched for putative members of this protein class in the CryptoDB database using as queries known Toxoplasma gondii Rhoptry molecules. We cloned a C. parvum sporozoite cDNA of 4269 bp encoding the sushi domain-containing protein cgd8_2530, which shared low amino acid sequence identity, yet a highly conserved domain architecture with the Rhoptry neck proteins TgRON1 of T. gondii and PfASP of Plasmodium falciparum . On denaturing and native gels, cgd8_2530 migrated at approximately 150 and 1000 kDa, respectively, suggesting an involvement in a multi-subunit protein complex. Immunoflorescence localised cgd8_2530 to a single, elongated area anterior to sporozoite micronemes and showed protein relocation to the parasite–host cell interface in early epicellular stages. Our data strongly suggest a Rhoptry localization for the newly characterised protein, which was therefore renamed C. parvum putative Rhoptry protein-1 (CpPRP1).
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Molecular dissection of novel trafficking and processing of the Toxoplasma gondii Rhoptry metalloprotease toxolysin-1.
Traffic (Copenhagen Denmark), 2011Co-Authors: Bettina E. Hajagos, Jean-françois Dubremetz, Stephen J Cheng, Jay M. Turetzky, Eric D. Peng, Christopher M. Ryan, Puneet Souda, Julian P. Whitelegge, Maryse Lebrun, Peter J BradleyAbstract:Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many Rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted Rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest Rhoptry targeting domain yet reported and expanded the consensus sequence of the Rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma Rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to Rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.
Ross L Coppel - One of the best experts on this subject based on the ideXlab platform.
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plasmodium Rhoptry proteins why order is important
Trends in Parasitology, 2013Co-Authors: Natalie A Counihan, Ming Kalanon, Ross L Coppel, Tania F De KoningwardAbstract:Apicomplexan parasites, including the Plasmodium species that cause malaria, contain three unusual apical secretory organelles (micronemes, rhoptries, and dense granules) that are required for the infection of new host cells. Because of their specialized nature, the majority of proteins secreted from these organelles are unique to Apicomplexans and are consequently poorly characterized. Although Rhoptry proteins of Plasmodium have been implicated in events central to invasion, there is growing evidence to suggest that proteins originating from this organelle play key roles downstream of parasite entry into the host cell. Here we discuss recent work that has advanced our knowledge of Rhoptry protein trafficking and function, and highlight areas of research that require further investigation.
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Dissecting the apicomplexan Rhoptry neck proteins.
Trends in parasitology, 2010Co-Authors: Nicholas Ian Proellocks, Ross L Coppel, Karena L. WallerAbstract:Apicomplexan parasites possess specialized secretory organelles (rhoptries and micronemes) that release their contents during host cell invasion. Although the rhoptries were once thought to be merely a bulbous ‘protein reservoir' connected to an anterior neck region, the localization of a protein specifically to the neck suggested that this region was more than just a duct. Recent studies have shown that the Rhoptry neck sub-compartment possesses a distinct protein repertoire. Some of these proteins share common features, including conservation across the phylum and involvement in tight-junction formation. A sub-group of Rhoptry neck proteins, the RONs, their association with the microneme protein apical membrane antigen AMA1, and their involvement in invasion are discussed.
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Identification of Rhoptry trafficking determinants and evidence for a novel sorting mechanism in the malaria parasite Plasmodium falciparum.
PLoS pathogens, 2009Co-Authors: Dave Richard, Lev Kats, Christine Langer, Casilda G Black, Khosse Mitri, Justin A Boddey, Alan F. Cowman, Ross L CoppelAbstract:The Rhoptry of the malaria parasite Plasmodium falciparum is an unusual secretory organelle that is thought to be related to secretory lysosomes in higher eukaryotes. Rhoptries contain an extensive collection of proteins that participate in host cell invasion and in the formation of the parasitophorous vacuole, but little is known about sorting signals required for Rhoptry protein targeting. Using green fluorescent protein chimeras and in vitro pull-down assays, we performed an analysis of the signals required for trafficking of the Rhoptry protein RAP1. We provide evidence that RAP1 is escorted to the Rhoptry via an interaction with the glycosylphosphatidyl inositol-anchored Rhoptry protein RAMA. Once within the Rhoptry, RAP1 contains distinct signals for localisation within a sub-compartment of the organelle and subsequent transfer to the parasitophorous vacuole after invasion. This is the first detailed description of Rhoptry trafficking signals in Plasmodium.
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Plasmodium falciparum Pf34, a novel GPI-anchored Rhoptry protein found in detergent-resistant microdomains.
International journal for parasitology, 2007Co-Authors: Nicholas Ian Proellocks, Lev Kats, Casilda G Black, Svetozar Kovacevic, David J. P. Ferguson, Belinda Joan Morahan, Karena L. Waller, Ross L CoppelAbstract:Abstract Apicomplexan parasites are characterised by the presence of specialised organelles, such as rhoptries, located at the apical end of invasive forms that play an important role in invasion of the host cell and formation of the parasitophorous vacuole. In this study, we have characterised a novel Plasmodium falciparum Rhoptry protein, Pf34, encoded by a single exon gene located on chromosome 4 and expressed as a 34 kDa protein in mature asexual stage parasites. Pf34 is expressed later in the life cycle than the previously described Rhoptry protein, Rhoptry Associated Membrane Antigen (RAMA). Orthologues of Pf34 are present in other Plasmodium species and a potential orthologue has also been identified in Toxoplasma gondii . Indirect immunofluorescence assays show that Pf34 is located at the merozoite apex and localises to the Rhoptry neck. Pf34, previously demonstrated to be glycosyl-phosphatidyl-inositol (GPI)-anchored [Gilson, P.R., Nebl, T., Vukcevic, D., Moritz, R.L., Sargeant, T., Speed, T.P., Schofield, L., Crabb, B.S. (2006) Identification and stoichiometry of GPI-anchored membrane proteins of the human malaria parasite Plasmodium falciparum . Mol. Cell. Proteomics 5, 1286–1299.], is associated with parasite-derived detergent-resistant microdomains (DRMs). Pf34 is carried into the newly invaded ring, consistent with a role for Pf34 in the formation of the parasitophorous vacuole. Pf34 is exposed to the human immune system during infection and is recognised by human immune sera collected from residents of malaria endemic areas of Vietnam and Papua New Guinea.
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Plasmodium rhoptries: how things went pear-shaped.
Trends in parasitology, 2006Co-Authors: Lev Kats, Casilda G Black, Nicholas Ian Proellocks, Ross L CoppelAbstract:Plasmodium parasites have three sets of specialised secretory organelles at the apical end of their invasive forms – rhoptries, micronemes and dense granules. The contents of these organelles are responsible for or contribute to host cell invasion and modification, and at least four apical proteins are leading vaccine candidates. Given the unusual nature of Plasmodium invasion, it is not surprising that unique proteins are involved in this process. Nowhere is this more evident than in rhoptries. We have collated data from several recent studies to compile a Rhoptry proteome. Discussion is focussed here on Rhoptry content and function.
Maryse Lebrun - One of the best experts on this subject based on the ideXlab platform.
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Assessing Rhoptry Secretion in T. gondii.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Catherine Suarez, Melissa B. Lodoen, Maryse LebrunAbstract:Rhoptries are key secretory organelles for Toxoplasma gondii invasion. Here, we describe how to assess the ability of T. gondii tachyzoites to secrete their Rhoptry contents in vitro.
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Identification of a new Rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii.
PloS one, 2012Co-Authors: Mauld H. Lamarque, Jean-françois Dubremetz, Gaelle Lentini, Julien Papoin, Anne-laure Finizio, Alexander W. Pfaff, Ermanno Candolfi, Maryse LebrunAbstract:Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains Rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains Rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of Rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
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identification of a new Rhoptry neck complex ron9 ron10 in the apicomplexa parasite toxoplasma gondii
PLOS ONE, 2012Co-Authors: Mauld H. Lamarque, Jean-françois Dubremetz, Gaelle Lentini, Julien Papoin, Anne-laure Finizio, Alexander W. Pfaff, Ermanno Candolfi, Maryse LebrunAbstract:Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains Rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains Rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of Rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
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Molecular dissection of novel trafficking and processing of the Toxoplasma gondii Rhoptry metalloprotease toxolysin-1.
Traffic (Copenhagen Denmark), 2011Co-Authors: Bettina E. Hajagos, Jean-françois Dubremetz, Stephen J Cheng, Jay M. Turetzky, Eric D. Peng, Christopher M. Ryan, Puneet Souda, Julian P. Whitelegge, Maryse Lebrun, Peter J BradleyAbstract:Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many Rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted Rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest Rhoptry targeting domain yet reported and expanded the consensus sequence of the Rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma Rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to Rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.
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Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol
Biochemical Journal, 2008Co-Authors: Sébastien Besteiro, Henri Vial, Maryse Lebrun, Justine Bertrand-michel, Jean-françois DubremetzAbstract:Rhoptries are secretory organelles involved in the virulence of the human pathogen Toxoplasma gondii. We have used high performance liquid chromatography and capillary gas-liquid chromatography to isolate and quantify lipids from whole Toxoplasma cells and their purified rhoptries. This comparative lipidomic analysis revealed an enrichment of cholesterol, sphingomyelin and, most of all, saturated fatty acids in the rhoptries. These lipids are known, when present in membranes, to be contributing to their rigidity and, interestingly, fluorescence anisotropy measurements confirmed that Rhoptry-derived membranes have a lower fluidity than membranes from whole T. gondii cells. Moreover, while rhoptries were initially thought to be highly enriched in cholesterol, we demonstrated it is present in lower proportions and provided additional evidence towards a lack of involvement of Rhoptry cholesterol in the process of host cell invasion by the parasite. Indeed, depleting the cholesterol content of the parasites did not prevent the secretion of protein-containing Rhoptry-derived vesicles and the parasites could still establish a structure called the moving junction, which is necessary for invasion. Instead, the crucial role for host cholesterol for invasion, which has already been demonstrated (Coppens, I. and Joiner, K. A. (2003), Mol.Biol.Cell 14, 3804-3820), might be explained by the need of a cholesterol-rich region of the host cell we could visualise at the point of contact with the attached parasite, in conditions where parasite motility was blocked.
Manuel A. Patarroyo - One of the best experts on this subject based on the ideXlab platform.
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PvRON2, a new Plasmodium vivax Rhoptry neck antigen
Malaria journal, 2011Co-Authors: Gabriela Arévalo-pinzón, Hernando Curtidor, Liliana C Patiño, Manuel A. PatarroyoAbstract:Background Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within Rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively.
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Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue.
Biochemical and biophysical research communications, 2007Co-Authors: Alvaro Mongui, Oscar Perez-leal, Jose Rojas-caraballo, Diana I. Angel, Jimena Cortés, Manuel A. PatarroyoAbstract:Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a Rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of Rhoptry proteins.
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The Plasmodium vivax Rhoptry-associated protein 1.
Biochemical and biophysical research communications, 2006Co-Authors: Oscar Perez-leal, Alvaro Mongui, Jimena Cortés, Gloria Yepes, Jesus Leiton, Manuel A. PatarroyoAbstract:Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum Rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' Rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its Rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.
John C. Boothroyd - One of the best experts on this subject based on the ideXlab platform.
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translocation of dense granule effectors across the parasitophorous vacuole membrane in toxoplasma infected cells requires the activity of rop17 a Rhoptry protein kinase
mSphere, 2019Co-Authors: Michael W Panas, Abel Ferrel, Adit Naor, Elizabeth Tenborg, Hernan Lorenzi, John C. BoothroydAbstract:ABSTRACT Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of Rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for Rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known Rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction. IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite Rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.
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translocation of dense granule effectors across the parasitophorous vacuole membrane in toxoplasma infected cells requires the activity of rop17 a Rhoptry protein kinase
bioRxiv, 2019Co-Authors: Michael W Panas, Abel Ferrel, Adit Naor, Elizabeth Tenborg, Hernan Lorenzi, John C. BoothroydAbstract:Abstract Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of Rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for Rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing 3 components, MYR1, MYR2 and MYR3. To determine if other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known Rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host-cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and, thereby, inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM, but that translocation can be rescued “in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host-cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction. Importance When Toxoplasma infects a cell it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite Rhoptry protein, ROP17, is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins thereby preventing Toxoplasma from productively infecting its animal and human hosts.
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4-Bromophenacyl bromide specifically inhibits Rhoptry secretion during Toxoplasma invasion.
PloS one, 2009Co-Authors: Sandeep Ravindran, Melissa B. Lodoen, Steven H. L. Verhelst, Matthew Bogyo, John C. BoothroydAbstract:Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of Rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of Rhoptry secretion in Toxoplasma invasion. This drug specifically blocked Rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified “click-chemistry” forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and Rhoptry secretion, in particular.
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the toxoplasma gondii dense granule protein gra7 is phosphorylated upon invasion and forms an unexpected association with the Rhoptry proteins rop2 and rop4
Infection and Immunity, 2008Co-Authors: Joe Dan Dunn, Sandeep Ravindran, Seonkyeong Kim, John C. BoothroydAbstract:The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain Rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the Rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.
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Plasmodium falciparum AMA1 Binds a Rhoptry Neck Protein Homologous to TgRON4, a Component of the Moving Junction in Toxoplasma gondii
Eukaryotic cell, 2006Co-Authors: David L Alexander, Jean-françois Dubremetz, Shirin Arastu-kapur, John C. BoothroydAbstract:Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted Rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the Rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.