The
            <i>Toxoplasma gondii</i>
            Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4

Dunn, Joe Dan; Ravindran, Sandeep; Kim, Seon Kyeong; Boothroyd, John C.

doi:10.1128/iai.01667-07

Cited by 84 publications

(80 citation statements)

References 37 publications

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“…S1). Two peptides of ROP4/7 were also identified in the ROP18-Ty immunoprecipitation (IP), which may reflect the previously described interaction between GRA7 and ROP4/7 (23). To validate the ROP18 binding partners identified by MS/MS, GRA7 was immunoprecipitated from WT RH strain vs. Δgra7 mutant parasites under similar conditions and analyzed by MS/MS.…”

Section: Significancementioning

confidence: 78%

“…Previous co-IPs also identified an interaction between the pseudokinases ROP4/7, ROP8/2, and GRA7 and demonstrated that these proteins become phosphorylated within the host cell (23). Deletion of either GRA7 or ROP8/2 alone did not decrease virulence, unlike the loss of ROP18, suggesting that GRA7 and ROP8/2 are not involved in regulating ROP18 function.…”

Section: Discussionmentioning

confidence: 95%

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Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

Alaganan

Fentress

Tang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

115

150

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The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant Δgra7 or Δrop18 was partially attenuated for virulence in mice, whereas the combined Δgra7Δrop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.pathogenesis | innate immunity | cooperative polymerization T he apicomplexan parasite Toxoplasma gondii has a remarkable host range and is capable of infecting most warm-blooded animals (1). The cellular life cycle of this opportunistic pathogen involves active invasion of nucleated host cells and establishment of an intracellular niche within the parasitophorous vacuole. This compartment is demarcated by the parasitophorous vacuole membrane (PVM), which avoids fusion with the host endomembrane system, although being studded with many parasite proteins (2). PVM-localized proteins derive from two major organelles: the rhoptries (ROP proteins) and the dense granules (GRA proteins), which are sequentially secreted upon invasion (3). The strategic location of GRAs and ROPs on the PVM positions them to play important roles in interacting with the host.ROP proteins are secreted directly into the host cell cytosol at the time of invasion, after which they target to the PVM or other locations within the cell (4). Although many ROP proteins contain a kinase fold, nearly half of these are predicted to be pseudokinases because they lack the critical catalytic residues that are normally required for phosphate transfer (5). Often, these pseudokinases (i.e., ROP5, ROP8/2, ROP4/7) exist as tandem gene duplications and show evidence of positive selection (5). Following secretion, ROP2 family members are targeted to the cytoplasmic face of the PVM via a series of amphipathic α-helical regions in their N termini (6, 7).ROP proteins generated renewed interest when it became evident that some ROPs confer critical strain-specific virulence in mice (8-12). In particular, the active kinase ROP18 and the pseudokinase ROP5 defend the parasite vacuole by blocking the ...

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Section: Significancementioning

confidence: 78%

Section: Discussionmentioning

confidence: 95%

Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

Alaganan

Fentress

Tang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

115

150

View full text Add to dashboard Cite

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“…2C). A fraction of GRA7 is associated with the vacuolar network of tubular membranes rather than the PVM (Bonhomme et al ., 1998), and in addition, binding of GRA7 to ROP2 and ROP4 was shown (Dunn et al ., 2008), perhaps explaining the only partial decrease of vacuolar GRA7 amounts in the absence of ROP5.…”

Section: Discussionmentioning

confidence: 99%

“…Yet, both phosphorylation and glycosylation have been reported for members of the ROP5/ROP18/GRA7 complex (Dunn et al ., 2008; Luo et al ., 2011; Treeck et al ., 2011). It is possible that some of these modifications may be involved in the interactions between components of the kinase complex.…”

Section: Discussionmentioning

confidence: 99%

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

Hermanns

Müller

Könen‐Waisman

et al. 2015

Cellular Microbiology

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SummaryIn mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.

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“…All subsequent incubations were performed in phosphate-buffered saline (PBS) supplemented with 3% bovine serum albumin (BSA). The cells were permeabilized for 15 min with PBS supplemented with 0.2% Triton X-100 and subsequently were blocked in 3% BSA-PBS overnight at 4°C or at room temperature for 1 h. After incubation with the primary antibody, Alexa Fluor 594-and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes/Invitrogen) were used (25). Samples were viewed on an Olympus BX60 upright fluorescence microscope with a 100ϫ oil immersion lens, and images were acquired with Image-Pro Plus software or QCapture software (version 2.9.11.2).…”

Section: Methodsmentioning

confidence: 99%

GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

et al. 2014

Self Cite

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The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between type II and type III Toxoplasma strains and quantitative trait locus (QTL) mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside the parasites and is found within the parasitophorous vacuole. In vitro experiments with a type II ⌬gra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 than those infected with the wild-type or complemented control parasites. In vivo experiments showed that mice infected with a type II ⌬gra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites and that complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the type II and type III versions of GRA25 differ in endogenous expression levels; however, both are able to promote parasite expansion in vivo when expressed in a type II ⌬gra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.T oxoplasma gondii is an obligate intracellular parasite with the remarkable ability to infect a broad range of mammalian and avian hosts. During infection in the mouse, a natural Toxoplasma host, macrophages are known to play a key role in mounting an immune response to infection via the secretion of cytokines such as interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-␣) (1). The secretion of these cytokines leads to the activation of NK cells and CD4 ϩ and CD8 ϩ T cells (2-4). Although macrophages can themselves be targets of parasite infection, they also can control parasite dissemination through cell-intrinsic, antimicrobial strategies, such as the production of nitric oxide (NO) and reactive oxygen species (ROS) and, in mice, the activation of immunityrelated GTPases (IRGs) (5, 6). Recent studies have shown that inflammatory monocytes, in particular, are recruited to the site of infection by the cytokine CCL2 (monocyte chemotactic protein-1 [MCP-1]) (7, 8). As has been described for infection with several pathogens (9), CCL2 is essential for control of parasite spread and survival of infection in both intraperitoneal (i.p.) and oral Toxoplasma infections (7,8).The strains of Toxoplasma that predominate in Europe and North America, termed types I, II, and III, differ in a wide range of phenotypes, including how they interface with the immune response. The tachyzoite stage of the parasite interacts with the host through secretion of polymorphic proteins from specialized organelles termed rhoptries, which house "ROP" proteins, and dense granules, which contain "GRA" proteins. One such polymorphic protein is the tyrosine kinase ROP16. Upon injection into the host cell, it directly phosphorylates STAT3 and STAT6, and in cells infec...

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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

Cited by 84 publications

References 37 publications

Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

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