Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii

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.

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

10.1101/2021.11.02.467042 ◽

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.

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The Toxoplasma gondii Cyst Wall Interactome

mBio ◽

10.1128/mbio.02699-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Vincent Tu ◽

Tadakimi Tomita ◽

Tatsuki Sugi ◽

Joshua Mayoral ◽

Bing Han ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Interactions ◽

Cyst Wall ◽

Affinity Purification ◽

Dense Granule ◽

Hypothetical Proteins ◽

Content Type

ABSTRACT A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

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Faculty Opinions recommendation of Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8311986.8782056 ◽

2011 ◽

Author(s):

Jonathan Howard ◽

Julia P Hunn

Keyword(s):

Toxoplasma Gondii ◽

Dense Granule ◽

Granule Protein ◽

Nf Kappab

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The amino-terminal region of dense granule protein 6 of Toxoplasma gondii stimulates IFN-γ production by microglia

Microbes and Infection ◽

10.1016/j.micinf.2019.12.003 ◽

2020 ◽

Vol 22 (8) ◽

pp. 375-378 ◽

Cited By ~ 1

Author(s):

Qila Sa ◽

Corinne Mercier ◽

Marie-France Cesbron-Delauw ◽

Yasuhiro Suzuki

Keyword(s):

Toxoplasma Gondii ◽

Dense Granule ◽

Terminal Region ◽

Amino Terminal ◽

Granule Protein ◽

Ifn Γ

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First Characterization of theNeospora caninumDense Granule Protein GRA9

BioMed Research International ◽

10.1155/2017/6746437 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 5

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.

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