scholarly journals Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein

2011 ◽  
Vol 208 (1) ◽  
pp. 195-212 ◽  
Author(s):  
Emily E. Rosowski ◽  
Diana Lu ◽  
Lindsay Julien ◽  
Lauren Rodda ◽  
Rogier A. Gaiser ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Nuclear Translocation ◽  
Strain Differences ◽  
Dense Granule ◽  
Forward Genetics ◽  
Type I ◽  
Type Ii ◽  
Granule Protein

NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB–mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii’s host cell–modifying arsenal.

scholarly journals A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation

2013 ◽  
Vol 210 (10) ◽  
pp. 2071-2086 ◽  
Author(s):  
Laurence Braun ◽  
Marie-Pierre Brenier-Pinchart ◽  
Manickam Yogavel ◽  
Aurélie Curt-Varesano ◽  
Rose-Laurence Curt-Bertini ◽  
...  
Keyword(s):  
Immune Response ◽  
Host Cell ◽  
Nuclear Translocation ◽  
Parasitophorous Vacuole ◽  
Structural Features ◽  
Mapk Signaling ◽  
Dense Granule ◽  
Interleukin 12 ◽  
Granule Protein

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24–p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.

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 Dense granule protein, GRA64 interacts with host cell ESCRT proteins during Toxoplasma gondii infection

2021 ◽  
Author(s):  
Joshua Mayoral ◽  
Rebekah B. Guevara ◽  
Yolanda Rivera-Cuevas ◽  
Vincent Tu ◽  
Tadakimi Tomita ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Electron Tomography ◽  
Cell Activity ◽  
Dense Granule ◽  
Secreted Proteins ◽  
Toxoplasma Infection ◽  
Granule Protein ◽  
Novel Host ◽  
Toxoplasma Gondii Infection

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In attempts to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a trans-membrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrate that GRA64 appears to interact with certain components of the host Endosomal Sorting Complexes Required for Transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence in mice nor encystation as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Dgra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection.

scholarly journals Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15

2018 ◽  
Vol 117 (9) ◽  
pp. 2785-2793 ◽  
Author(s):  
Qing Liu ◽  
Wen-Wei Gao ◽  
Hany M. Elsheikha ◽  
Jun-Jun He ◽  
Fa-Cai Li ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Response ◽  
Dense Granule ◽  
Transcriptomic Analysis ◽  
Host Cell Response ◽  
Granule Protein

scholarly journals Toxoplasma gondiiParasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Barbara A. Fox ◽  
Rebekah B. Guevara ◽  
Leah M. Rommereim ◽  
Alejandra Falla ◽  
Valeria Bellini ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Gamma Interferon ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Vacuole Membrane ◽  
Ifn Γ

ABSTRACTToxoplasma gondiievades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronicToxoplasmainfection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cystsin vivowithout affecting the parasite growth rate or the ability to differentiate into cystsin vitro. Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4mutants was reduced but not abolished. In contrast, the PruΔgra12mutant was avirulent in mice and PruΔgra12parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12and type II PruΔgra12parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80and type II PruΔku80strains, respectively. Despite this resistance, Δgra12PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondiicysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cystsin vivo. A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.

scholarly journals Type II Toxoplasma gondii Induction of CD40 on Infected Macrophages Enhances Interleukin-12 Responses

2014 ◽  
Vol 82 (10) ◽  
pp. 4047-4055 ◽  
Author(s):  
Pedro Morgado ◽  
Dattanand M. Sudarshana ◽  
Lanny Gov ◽  
Katherine S. Harker ◽  
Tonika Lam ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Transcript Level ◽  
Forward Genetics ◽  
Interleukin 12 ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Content Type ◽  
Type Iii ◽  
Infected Cells

ABSTRACTToxoplasma gondiiis an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controllingT. gondiiinfection. We examined the regulation of CD40 on the surface ofT. gondii-infected bone marrow-derived macrophages (BMdMs).T. gondiiinduced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type IIT. gondii-infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele ofGRA15(GRA15II), we found that type I GRA15IIparasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15IIin THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15IIinduction of CD40 promotes parasite immunity through the production of IL-12.

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.

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.

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