Affinage · Toxoplasma gondii

GRA16

dense granule protein GRA16 · UniProt A0A125YUV5

Annotated
2026-09-05
29 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GRA16 is a Toxoplasma gondii dense granule effector that is exported across the parasitophorous vacuole membrane (PVM) into the host cell, where it reaches the nucleus and reprograms host-cell physiology to favor parasite virulence and immune evasion (PMID:23601110). Export is a multistep, tightly gated process: GRA16 must first be cleaved at an N-terminal PEXEL-like (RRLAE) motif by the Golgi aspartyl protease TgASP5, without which it is retained in the vacuolar space, and export additionally requires a second N-terminal element (PMID:26270241, PMID:26473595); it must also be dephosphorylated by the secreted parasite phosphatase TgPPM3C, since phosphomimetic mutation of two serine residues blocks export (PMID:33370417). Trans-PVM translocation then proceeds through the MYR translocon, requiring MYR1, MYR2, MYR3, MYR4, GRA44, and GRA45, consistent with passage of the unfolded effector through a channel (PMID:26838724, PMID:29357375, PMID:32075880). In the host nucleus, GRA16 binds the deubiquitinase HAUSP and PP2A phosphatase to modulate the p53 pathway and cell-cycle gene expression, and it is the effector responsible for Toxoplasma's upregulation of host c-Myc (PMID:23601110, PMID:32581080). Through these host targets GRA16 also inhibits NF-κB activation via PP2A-B55 and contributes, together with other MYR-dependent effectors, to blocking the IFNγ-driven host response and to CCR7-mediated macrophage hypermigration (PMID:32927892, PMID:39292011, PMID:39207098). Export is stage-specific, occurring in tachyzoites but not in bradyzoite tissue cysts (PMID:30406043).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2013 High

    Established that a dense granule protein can act as a nuclear-targeting host effector, defining GRA16 as an exported modulator of host cell-cycle and p53 pathways with defined host-enzyme partners.

    Evidence Reciprocal co-IP for HAUSP and PP2A binding, imaging/fractionation for nuclear localization, and GRA16-deficient strain phenotyping

    PMID:23601110

    Open questions at the time
    • Export machinery not yet identified
    • Structural basis of HAUSP/PP2A binding unresolved
    • Direct link between p53 modulation and virulence not dissected
  2. 2015 High

    Identified the proteolytic gate for export, showing GRA16 is matured by TgASP5 cleavage at a PEXEL-like motif and that this cleavage plus a second N-terminal element are required to escape the vacuole.

    Evidence Dual N-/C-terminal epitope-tag proteolysis assays, asp5 knockout/complementation, and N-terminal domain mutagenesis with immunofluorescence

    PMID:26270241 PMID:26473595

    Open questions at the time
    • Identity/function of the second required N-terminal element unresolved
    • How cleavage licenses translocation mechanistically unknown
  3. 2016 High

    Defined the trans-PVM translocation requirement, establishing that GRA16 nuclear delivery depends on MYR1 at the PVM.

    Evidence myr1 genetic deletion with immunofluorescence tracking of GRA16 nuclear localization and transcriptome screening

    PMID:26838724

    Open questions at the time
    • Composition of the full translocon unknown at this stage
    • Whether MYR1 forms a pore not established
  4. 2018 High

    Expanded the translocon to a multi-subunit machinery and provided evidence for a channel-based mechanism requiring effector unfolding.

    Evidence MYR2/MYR3 knockouts, MYR1/MYR3 co-IP, and a DHFR structural-fusion transport-block assay

    PMID:29357375

    Open questions at the time
    • Direct demonstration of a pore structure lacking
    • Energetics/driving force of translocation unknown
  5. 2018 Medium

    Showed that GRA16 export is developmentally restricted, occurring in tachyzoites but not bradyzoite tissue cysts, implying stage-specific host manipulation.

    Evidence Tetracycline-inducible HA-tagged GRA16 after bradyzoite conversion with immunofluorescence localization

    PMID:30406043

    Open questions at the time
    • Single-method, single-lab observation
    • Mechanism restricting export in cysts unknown
  6. 2019 Medium

    Refined the p53 mechanism, showing HAUSP-bound GRA16 stabilizes p53 via nuclear PTEN rather than MDM2, driving p53-dependent apoptosis.

    Evidence Stable GRA16 expression in p53-WT HepG2 vs p53-null Hep3B cells, HAUSP co-IP, nuclear fractionation, and xenograft models

    PMID:30834688

    Open questions at the time
    • No mutagenesis of GRA16 residues required for HAUSP binding
    • Relevance to natural infection context untested
  7. 2020 High

    Completed the translocon parts list and linked it to a defined GRA16-driven host phenotype, showing GRA44/GRA45/MYR4 are needed for c-Myc/cyclin E1 upregulation.

    Evidence MYR1 affinity purification, knockouts of GRA44/GRA45/MYR4, GRA16 localization, and host c-Myc/cyclin E1 assays

    PMID:32075880

    Open questions at the time
    • Stoichiometry/architecture of the assembled translocon unresolved
    • Direct GRA16 role in c-Myc induction not separated from translocation defect
  8. 2020 High

    Assigned host c-Myc upregulation specifically to GRA16 via gain- and loss-of-function, establishing it as the responsible effector.

    Evidence Heterologous GRA16 expression in Neospora caninum and gra16 deletion in Toxoplasma with c-Myc readouts

    PMID:32581080

    Open questions at the time
    • Molecular route from GRA16 to c-Myc transcription undefined
    • Whether HAUSP/PP2A binding mediates c-Myc effect unknown
  9. 2020 High

    Added a dephosphorylation checkpoint to export, showing TgPPM3C dephosphorylates GRA16 and that this is selectively required for its export.

    Evidence pppm3c knockout, vacuolar phosphoproteomics, and GRA16 serine phosphomimetic mutagenesis with localization tracking

    PMID:33370417

    Open questions at the time
    • Why GRA16 specifically requires dephosphorylation while other effectors do not is unclear
    • Kinase that phosphorylates these sites not identified
  10. 2020 Medium

    Connected GRA16 to NF-κB suppression via a PP2A-B55/GWL/ENSA axis dampening AKT/ERK and IKKβ signaling.

    Evidence Stable GRA16 expression in H1299 cells with western-blot pathway dissection, siRNA/pharmacology, and xenograft model

    PMID:32927892

    Open questions at the time
    • Performed largely outside native infection context
    • Direct GRA16-PP2A-B55 interaction not shown at residue level
  11. 2024 Medium

    Placed GRA16 within a redundant effector set required to fully block the IFNγ response in human cells, protecting against premature egress and host cell death.

    Evidence Genome-wide loss-of-function screens in IFNγ-primed human fibroblasts, individual GRA knockouts, and egress/death assays with murine comparison

    PMID:39292011

    Open questions at the time
    • GRA16's isolated mechanistic contribution not separated from the other three effectors
    • Human-vs-murine specificity mechanism unknown
  12. 2024 Medium

    Implicated GRA16 as a contributor to CCR7-driven macrophage hypermigration and dissemination alongside other effectors.

    Evidence GRA effector deletion mutants with CCR7 and macrophage chemotaxis assays and in vivo adoptive-transfer migration

    PMID:39207098

    Open questions at the time
    • GRA16 described as contributory, not primary driver
    • Downstream target linking GRA16 to CCR7 unidentified
  13. 2024 Medium

    Repurposed the GRA16 export system as a biotechnology tool, demonstrating nuclear delivery of large therapeutic cargo including into neurons in vivo.

    Evidence GRA16 translational fusions (e.g. GRA16-MeCP2), pull-downs, brain organoids, scRNA-seq, and in vivo intraperitoneal delivery with brain imaging

    PMID:39075233

    Open questions at the time
    • Efficiency and safety of delivery not fully characterized
    • Whether native export determinants limit cargo size undefined
  14. 2026 Low

    Positioned GRA16 as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-κB pathway via TRAF3 depletion and NIK/p100 processing.

    Evidence Combinatorial GRA effector knockout panels with RelB/p52/TRAF3/NIK/p100 readouts in human and murine cells (preprint)

    PMID:41959509

    Open questions at the time
    • Preprint; GRA16's individual contribution inferred additively, not shown in isolation
    • Apparent tension with reported GRA16-mediated NF-κB suppression not reconciled

Open questions

Synthesis pass · forward-looking unresolved questions
  • How GRA16's host targets (HAUSP, PP2A) mechanistically integrate its multiple downstream effects (p53, c-Myc, NF-κB, IFNγ protection, migration) into a single coherent virulence program remains unresolved.
  • No structural model of GRA16 or its host-partner interfaces
  • Residue-level mapping of HAUSP/PP2A binding lacking
  • Divergent NF-κB outcomes (suppression vs non-canonical activation) not reconciled

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
HAUSP (HOST DEUBIQUITINASE)MYR1 (TRANSLOCON COMPONENT, EXPORT)PP2A HOLOENZYME / PP2A-B55 REGULATORY SUBUNIT (HOST PHOSPHATASE)TGASP5 (PARASITE GOLGI ASPARTYL PROTEASE, PROCESSING)TGPPM3C (PARASITE PHOSPHATASE, EXPORT REGULATION)
Complex memberships
MYR translocon (MYR1/MYR2/MYR3/MYR4/GRA44/GRA45) mediating trans-PVM effector export

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2013 GRA16 is a dense granule protein that is exported through the parasitophorous vacuole membrane (PVM) and traffics to the host cell nucleus, where it positively modulates host genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 physically binds two host enzymes: the deubiquitinase HAUSP and PP2A phosphatase. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Co-immunoprecipitation (binding to HAUSP and PP2A), fluorescence/subcellular fractionation (nuclear localization), gene expression analysis, GRA16-deficient strain phenotyping (attenuated virulence) Cell host & microbe High 23601110
2015 TgASP5, a Golgi-resident aspartyl protease homologous to Plasmodium Plasmepsin V, cleaves GRA16 at its PEXEL-like (RRLAE) motif in the N-terminal region, and this cleavage is essential for GRA16 export beyond the PVM and trafficking to the host cell nucleus. In the absence of ASP5, GRA16 is retained within the vacuolar space. N- and C-terminal epitope tagging demonstrating proteolysis; asp5 knockout/conditional mutants showing retention of GRA16 in vacuole; immunofluorescence localization; genetic complementation Cellular microbiology High 26270241 26473595
2015 GRA16 export requires two protein elements in its N-terminal region: a PEXEL-like motif (RRLAE) required for TgASP5-mediated cleavage, and a second element also necessary for export. TgASP5 directly mediates GRA16 maturation and export in a PEXEL-dependent manner. N- and C-terminal epitope-tag proteolysis assays; asp5 deletion mutants; domain deletion/mutation of GRA16 N-terminus; immunofluorescence Cellular microbiology High 26270241
2016 GRA16 export from the parasitophorous vacuole into the host cytosol/nucleus is dependent on MYR1, a protein at the PVM that forms part of an effector translocation machinery. In myr1 deletion mutants, GRA16 fails to reach the host cell nucleus. myr1 genetic deletion; immunofluorescence tracking of GRA16 nuclear localization in infected cells; flow cytometry-based screen for host transcriptome reprogramming mBio High 26838724
2018 GRA16 translocation across the PVM into the host cell requires MYR2 and MYR3 in addition to MYR1. MYR2 and MYR3 co-localize with MYR1 at the PVM and are membrane-associated. MYR3, but not MYR2, stably associates with MYR1. Fusing a structured DHFR domain to intrinsically disordered effectors blocks their transport, consistent with a translocon-based mechanism. Genetic deletion of MYR2/MYR3; immunofluorescence of GRA16 nuclear localization; co-immunoprecipitation of MYR1/MYR3; DHFR-fusion effector transport block assay PLoS pathogens High 29357375
2020 Three additional MYR1-interacting proteins—GRA44, GRA45, and MYR4—are essential for translocation of GRA16 into the host cell and for GRA16-dependent upregulation of human c-Myc and cyclin E1 in infected cells. These proteins were identified by affinity purification of MYR1. MYR1 affinity purification/co-immunoprecipitation; genetic knockout of GRA44, GRA45, MYR4; immunofluorescence of GRA16 nuclear localization; host c-Myc/cyclin E1 upregulation assay mSphere High 32075880
2020 GRA16 is responsible for Toxoplasma's upregulation of host c-Myc. Expression of GRA16 in Neospora caninum (which does not upregulate host c-Myc) conferred this ability. Deletion of GRA16 from Toxoplasma severely diminished host c-Myc upregulation. Heterologous expression of GRA16 in Neospora caninum; gra16 genetic deletion in Toxoplasma; flow cytometry/immunofluorescence for c-Myc levels mSphere High 32581080
2020 The secreted parasite phosphatase TgPPM3C regulates GRA16 export by dephosphorylating GRA16; phosphomimetic mutation of two GRA16 serine residues causes export defects, demonstrating that dephosphorylation of GRA16 is a critical step in its export process. Loss of TgPPM3C results in defective GRA16 export but not export of other MYR1-dependent effectors. pppm3c genetic deletion; phosphoproteomics of vacuolar proteins; phosphomimetic mutagenesis of GRA16 serine residues; immunofluorescence tracking of GRA16 nuclear localization PLoS pathogens High 33370417
2018 GRA16 and GRA24 are not secreted beyond the cyst wall membrane in bradyzoite (tissue cyst) stage Toxoplasma; using tetracycline-inducible HA-tagged expression after stage conversion, these proteins remained within cysts. This establishes that trans-PVM effector export by GRA16 is stage-specific and does not occur in tissue cysts. Tetracycline-inducible expression of HA-tagged GRA16 after bradyzoite induction; immunofluorescence localization Frontiers in cellular and infection microbiology Medium 30406043
2020 GRA16 inhibits NF-κB activation through upregulation of PP2A-B55 regulatory subunit in host cells. GRA16 expression directly elevated PP2A-B55 levels, decreased GWL protein levels and ENSA phosphorylation, activated PP2A-B55, and suppressed AKT/ERK phosphorylation, leading to NF-κB inactivation via reduced IKKβ, de-phosphorylation of IκBα, and reduced nuclear transit of NF-κB p65. Stable transfection of GRA16 in H1299 NSCLC cells; western blotting for PP2A-B55, GWL, ENSA, AKT, ERK, IKKβ, IκBα, NF-κB p65; xenograft mouse model; pharmacological and siRNA pathway dissection International journal of molecular sciences Medium 32927892
2019 HAUSP-bound GRA16 preferentially increases p53 stabilization via nuclear localization of PTEN rather than MDM2-dependent mechanisms in p53-wild-type HepG2 cells. GRA16 expression increased PTEN and p53 levels and induced apoptosis in a p53-dependent manner (absent in p53-null Hep3B cells). Stable GRA16 expression via retrovirus in HepG2 (p53-WT) and Hep3B (p53-null) cells; western blotting for PTEN, p53, MDM2, AKT; nuclear fractionation; xenograft mouse model; co-immunoprecipitation with HAUSP Journal of cellular and molecular medicine Medium 30834688
2024 GRA16, together with TgIST, GRA24, and GRA28, is necessary for a complete block of IFNγ response in human cells post-infection. Loss of GRA16 (among these four effectors) results in parasite premature egress and host cell death in IFNγ-stimulated human cells, a phenotype not observed in murine cells. This protection requires MYR translocon-dependent export of GRA16. Genome-wide loss-of-function screens in IFNγ-primed human fibroblasts; genetic KO of individual GRA effectors; parasite egress and host cell death assays across multiple human cell lines; comparison with murine cells mBio Medium 39292011
2024 GRA16 (alongside GRA18) contributes to CCR7-mediated macrophage hypermigration and dissemination of Toxoplasma, acting in concert with GRA15 (NF-κB pathway) and GRA24 (p38 MAPK pathway). GRA16/18 contributions were identified by effector-deletion experiments in macrophage chemotaxis assays. GRA effector deletion mutants; CCR7 expression and macrophage chemotaxis assays; in vivo adoptive transfer migration assay in mice mBio Medium 39207098
2024 GRA16 can be fused to large therapeutic proteins (>100 kDa) and used as a delivery vehicle to transport them into host cell nuclei, including neurons in vivo. As a proof of concept, GRA16-MeCP2 fusion was delivered to neurons via intraperitoneal administration in mice and distributed throughout the brain. Translational fusions to GRA16; immunofluorescence and pull-down assays in cultured cells and brain organoids; scRNA-seq; in vivo intraperitoneal administration and 3D brain distribution imaging in mice Nature microbiology Medium 39075233
2026 GRA16 is one of eight MYR1-dependent effectors (alongside IST, NSM, HCE1/TEEGR, GRA18, GRA24, GRA28, GRA84) that cooperate to activate the non-canonical NF-κB pathway in host cells. T. gondii infection drives depletion of TRAF3, stabilization of NIK, phosphorylation and processing of p100 to p52, and nuclear accumulation of RelB/p52. No single effector alone is responsible; the eight effectors act additively. Combinatorial knockout panels of GRA effectors; immunofluorescence and western blotting for RelB, p52, TRAF3, NIK, p100 in infected human and murine cells bioRxivpreprint Low 41959509

Source papers

Stage 0 corpus · 29 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression. Cell host & microbe 195 23601110
2016 A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells. mBio 133 26838724
2015 Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface. PLoS pathogens 95 26473595
2015 The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells. Cellular microbiology 84 26270241
2018 Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii. PLoS pathogens 75 29357375
2014 Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression. Cellular microbiology 54 24373221
2020 Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells. mSphere 53 32075880
2016 Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity. PLoS genetics 47 27447180
1999 The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental parasitology 46 10072322
2020 Toxoplasma GRA16 Inhibits NF-κB Activation through PP2A-B55 Upregulation in Non-Small-Cell Lung Carcinoma Cells. International journal of molecular sciences 37 32927892
2019 Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53-dependent apoptosis and anticancer effect. Journal of cellular and molecular medicine 26 30834688
1999 In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii. Parasitology 26 10503251
2018 Toxoplasma Does Not Secrete the GRA16 and GRA24 Effectors Beyond the Parasitophorous Vacuole Membrane of Tissue Cysts. Frontiers in cellular and infection microbiology 21 30406043
2024 Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons. Nature microbiology 20 39075233
2020 Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export. PLoS pathogens 20 33370417
2020 Toxoplasma Uses GRA16 To Upregulate Host c-Myc. mSphere 19 32581080
2022 PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 16 35810694
2024 Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination. mBio 13 39207098
2017 Resistance to Chronic Toxoplasma gondii Infection Induced by a DNA Vaccine Expressing GRA16. BioMed research international 13 28875149
2024 A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death. mBio 8 39292011
2025 Toxoplasma gondii GRA16 Suppresses Aerobic Glycolysis by Downregulating c-Myc and TERT Expressions in Colorectal Cancer Cells. Biomolecules & therapeutics 4 40556512
2025 Toxoplasma GRA16 attenuates Tau hyperphosphorylation and enhances autophagy in thrombin-treated HT-22 hippocampal neuronal cells. Scientific reports 2 40389494
2025 Proteome changes during in vitro culture adaptation of Toxoplasma gondii archetypal II and III field isolates. Frontiers in cellular and infection microbiology 1 41036225
2024 Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination. bioRxiv : the preprint server for biology 1 38370679
2023 A Combination of Four Nuclear Targeted Effectors Protects Toxoplasma Against Interferon Gamma Driven Human Host Cell Death During Acute Infection. bioRxiv : the preprint server for biology 1 38234811
2026 The Potential Function of Toxoplasma gondii in Tumour Immunotherapy. Immunology 0 42108599
2026 A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway. bioRxiv : the preprint server for biology 0 41959509
2019 Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell. mSphere 0 31366706
2015 Sequence variation and bioinformatics analysis of Toxoplasma gondii GRA16 Gene. Tropical biomedicine 0 26695219