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.

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.

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 Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

2014 ◽  
Vol 211 (10) ◽  
pp. 2013-2032 ◽  
Author(s):  
Ji Su Ma ◽  
Miwa Sasai ◽  
Jun Ohshima ◽  
Youngae Lee ◽  
Hironori Bando ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Dense Granule ◽  
Type I ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Host Immune Responses ◽  
Dense Granules ◽  
Granule Protein ◽  
C Terminus ◽  
Toxoplasma Gondii Infection

Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b+ Ly6G+ cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.

scholarly journals Secreted Effectors Modulating Immune Responses to Toxoplasma gondii

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 988
Author(s):  
Tadakimi Tomita ◽  
Rebekah B. Guevara ◽  
Lamisha M. Shah ◽  
Andrews Y. Afrifa ◽  
Louis M. Weiss
Keyword(s):  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Inflammatory Responses ◽  
Parasitophorous Vacuole ◽  
Congenital Infection ◽  
Dense Granule ◽  
Obligate Intracellular ◽  
Host Cytoplasm ◽  
Life Threatening ◽  
Intracellular Milieu

Toxoplasma gondii is an obligate intracellular parasite that chronically infects a third of humans. It can cause life-threatening encephalitis in immune-compromised individuals. Congenital infection also results in blindness and intellectual disabilities. In the intracellular milieu, parasites encounter various immunological effectors that have been shaped to limit parasite infection. Parasites not only have to suppress these anti-parasitic inflammatory responses but also ensure the host organism’s survival until their subsequent transmission. Recent advancements in T. gondii research have revealed a plethora of parasite-secreted proteins that suppress as well as activate immune responses. This mini-review will comprehensively examine each secreted immunomodulatory effector based on the location of their actions. The first section is focused on secreted effectors that localize to the parasitophorous vacuole membrane, the interface between the parasites and the host cytoplasm. Murine hosts are equipped with potent IFNγ-induced immune-related GTPases, and various parasite effectors subvert these to prevent parasite elimination. The second section examines several cytoplasmic and ER effectors, including a recently described function for matrix antigen 1 (MAG1) as a secreted effector. The third section covers the repertoire of nuclear effectors that hijack transcription factors and epigenetic repressors that alter gene expression. The last section focuses on the translocation of dense-granule effectors and effectors in the setting of T. gondii tissue cysts (the bradyzoite parasitophorous vacuole).

In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii

Parasitology ◽  
1999 ◽  
Vol 119 (3) ◽  
pp. 259-265 ◽  
Author(s):  
D. J. P. FERGUSON ◽  
D. JACOBS ◽  
E. SAMAN ◽  
J-F. DUBREMETZ ◽  
S. E. WRIGHT
Keyword(s):  
Toxoplasma Gondii ◽  
Staining Pattern ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasite Development ◽  
Double Labelling ◽  
Granule Protein ◽  
Specific Variation ◽  
Strong Staining

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

scholarly journals Toxoplasma gondiisalvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

2013 ◽  
Vol 24 (12) ◽  
pp. 1974-1995 ◽  
Author(s):  
Julia D. Romano ◽  
Sabrina Sonda ◽  
Emily Bergbower ◽  
Maria Elisa Smith ◽  
Isabelle Coppens
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
De Novo ◽  
Parasitophorous Vacuole ◽  
Parasite Growth ◽  
Sphingolipid Metabolism ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Parasite Replication

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

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 Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins

2021 ◽  
Author(s):  
Yolanda Rivera-Cuevas ◽  
Joshua Mayoral ◽  
Manlio Di Cristina ◽  
Anna-Lisa E. Lawrence ◽  
Einar B. Olafsson ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Cytosolic Proteins ◽  
Endosomal Sorting ◽  
Dense Granules ◽  
Escrt Machinery ◽  
Late Domain ◽  
Cytosolic Protein ◽  
Hiv Virus

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

scholarly journals Aspartyl Protease 5 matures virulence factors found at the host-parasite interface in Toxoplasma gondii

2018 ◽  
Author(s):  
Michael J Coffey ◽  
Laura F Dagley ◽  
Eugene A Kapp ◽  
Giuseppe Infusini ◽  
Justin A Boddey ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Virulence Factors ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Lytic Cycle ◽  
Aspartyl Protease ◽  
Protease Cleavage Sites ◽  
Granule Proteins ◽  
Host Parasite

AbstractToxoplasma gondii infects approximately 30% of the world’s population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection and several of these proteins are processed by the Golgi-associated Aspartyl Protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally-derived peptides from wildtype and Δasp5 parasites. We reveal over two thousand unique Toxoplasma N-terminal peptides, mapping to both natural N-termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterised ASP5-cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5, nor in wild type parasites following mutation of the motif from RRL⟶ARL. All new ASP5 substrates are dense granule proteins, and interestingly none appear to be exported, thus differing from the analogous system in related Plasmodium spp., instead revealing that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. Furthermore, we show that several of these ASP5-substrates are virulence factors, with their removal leading to attenuation in a mouse model, suggesting that phosphorylation at the host-parasite interface is important for virulence. Collectively, these data constitute the first in-depth analyses of the total list of ASP5 substrates, and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.

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