scholarly journals The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4

2008 ◽  
Vol 76 (12) ◽  
pp. 5853-5861 ◽  
Author(s):  
Joe Dan Dunn ◽  
Sandeep Ravindran ◽  
Seon-Kyeong Kim ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Opportunistic Pathogen ◽  
Dense Granule ◽  
Host Cells ◽  
Obligate Intracellular ◽  
Dense Granules ◽  
Obligate Intracellular Parasite ◽  
Granule Protein

ABSTRACT 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.

Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii

1995 ◽  
Vol 108 (4) ◽  
pp. 1669-1677 ◽  
Author(s):  
L.D. Sibley ◽  
I.R. Niesman ◽  
S.F. Parmley ◽  
M.F. Cesbron-Delauw
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Soluble Protein ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Host Cell Invasion ◽  
Cell Fractionation ◽  
Obligate Intracellular ◽  
Dense Granules ◽  
Storage Granules

Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the parasitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the parasitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.

scholarly journals Intervacuolar Transport and Unique Topology of GRA14, a Novel Dense Granule Protein in Toxoplasma gondii

2008 ◽  
Vol 76 (11) ◽  
pp. 4865-4875 ◽  
Author(s):  
Michael E. Rome ◽  
Josh R. Beck ◽  
Jay M. Turetzky ◽  
Paul Webster ◽  
Peter J. Bradley
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Obligate Intracellular ◽  
Dense Granules ◽  
Granule Protein ◽  
Host Cytoplasm ◽  
C Terminus ◽  
Membrane Bound ◽  
Host Parasite

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.

Invasion of Toxoplasma gondii occurs by active penetration of the host cell

1995 ◽  
Vol 108 (6) ◽  
pp. 2457-2464 ◽  
Author(s):  
J.H. Morisaki ◽  
J.E. Heuser ◽  
L.D. Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Tyrosine Phosphorylation ◽  
Host Cells ◽  
The Novel ◽  
Host Proteins ◽  
Host Cell Membrane ◽  
Membrane Ruffling ◽  
Obligate Intracellular ◽  
Obligate Intracellular Parasite

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25–40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2–4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.

scholarly journals Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Gustavo Arrizabalaga
Keyword(s):  
Host Cell ◽  
Protein Translocation ◽  
Parasitophorous Vacuole ◽  
Opportunistic Pathogen ◽  
Dense Granule ◽  
Effector Proteins ◽  
Content Type ◽  
Dense Granules ◽  
Cell Functions ◽  
Host Signaling

ABSTRACT The opportunistic pathogen Toxoplasma gondii is highly adept at manipulating host cell functions. While inside a host cell, Toxoplasma divides within a parasitophorous vacuole from which it secretes numerous effector proteins from its dense granules. Many of these so-called GRA proteins are translocated from the parsitophorous vacuole into the host cell where they directly disrupt host signaling pathways. The machinery that drives the translocation of GRA proteins across the parasitophorous vacuole membrane is being elucidated through both genetic and biochemical approaches. A new mSphere research article (M. W. Panas, A. Ferrel, A. Naor, E. Tenborg, et al., mSphere 4:e00276-19, 2019, https://doi.org/10.1128/mSphere.00276-19) describes how the kinase ROP17, which is secreted from the parasite’s rhoptries into the host cell during invasion, regulates the translocation of GRA effectors.

scholarly journals First Characterization of theNeospora caninumDense Granule Protein GRA9

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Margret Leineweber ◽  
Katrin Spekker-Bosker ◽  
Vanessa Ince ◽  
Gereon Schares ◽  
Andrew Hemphill ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Immunogold Electron Microscopy ◽  
Host Cell Invasion ◽  
Dense Granules ◽  
Granule Protein

The obligate intracellular apicomplexan parasiteNeospora caninum (N. caninum)is closely related toToxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of theN. caninumdense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the “excreted secreted antigen” (ESA) fraction ofN. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules ofN. caninum, and the truncated proteins show reduced secretion.

scholarly journals Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

2020 ◽  
Author(s):  
Suchita Rastogi ◽  
Yuan Xue ◽  
Stephen R. Quake ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Biology ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Transcriptomic Analysis ◽  
Host Cells ◽  
Intracellular Parasite ◽  
I Cells

ABSTRACTThe intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif

Parasitology ◽  
2005 ◽  
Vol 131 (2) ◽  
pp. 169-179 ◽  
Author(s):  
F. L. HENRIQUEZ ◽  
M. B. NICKDEL ◽  
R. MCLEOD ◽  
R. E. LYONS ◽  
K. LYONS ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Transmembrane Domain ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Type I ◽  
Dense Granules ◽  
Granule Protein

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory–secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine ‘KKXX’ endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

scholarly journals Transmembrane Insertion of the Toxoplasma gondii GRA5 Protein Occurs after Soluble Secretion into the Host Cell

1999 ◽  
Vol 10 (4) ◽  
pp. 1277-1287 ◽  
Author(s):  
Laurence Lecordier ◽  
Corinne Mercier ◽  
L. David Sibley ◽  
Marie-France Cesbron-Delauw
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Soluble Form ◽  
Host Cell Membrane ◽  
Dense Granules

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway inToxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

scholarly journals Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

2014 ◽  
Vol 13 (8) ◽  
pp. 965-976 ◽  
Author(s):  
Ira J. Blader ◽  
Anita A. Koshy
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Host Cells ◽  
Content Type ◽  
Obligate Intracellular ◽  
Cellular Processes ◽  
High Prevalence ◽  
Life Threatening ◽  
Cell Processes ◽  
Cellular Pathways

ABSTRACTIntracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections.Toxoplasma gondiiis an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence ofToxoplasmain humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by whichToxoplasmainteracts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.

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