GRA28

GRA28 is a Toxoplasma gondii dense granule effector that is secreted into the host cell nucleus via the MYR1 translocon, where it reprograms host gene expression to promote immune evasion and parasite dissemination (PMID:36309013, PMID:34781732). Its export from the vacuole depends on prior dephosphorylation by the secreted PP2C-class phosphatase TgPPM3C, and GRA28 export is defective in ΔTgPPM3C parasites (PMID:33370417). In the nucleus, GRA28 cooperates with host chromatin remodelers, boosting chromatin accessibility at the Ccr7 locus and enhancing GRA15/GRA24/NF-κB-dependent transcription to drive CCR7 upregulation and confer dendritic cell-like hypermigratory properties on parasitized macrophages (PMID:36309013, PMID:39207098, PMID:38370679). GRA28 is also required for T. gondii-induced CCL22 chemokine production across human and mouse immune and placental cells, and its deletion impairs parasite dissemination in vivo (PMID:34781732). As one of several nuclear MYR1-dependent effectors, GRA28 disrupts the histone acetyltransferase activity of CBP/p300 and influences IFNγ-driven transcription, contributing to blockade of IFNγ-driven host cell death (PMID:39292011, PMID:38234811). Within the effector network, GRA28 acts additively with other MYR1-dependent effectors to activate the non-canonical NF-κB (RelB/p52) pathway (PMID:41959509). In the virulent RH strain, GRA28 deletion has no effect on in vitro growth, egress, or acute virulence, indicating its dissemination and immune-modulatory roles are separable from acute lytic fitness (PMID:30211128).

1. 2018 Medium

   Whether GRA28 contributes to core lytic fitness was unknown; deleting it in the virulent RH strain established that GRA28 is dispensable for in vitro growth, egress, and acute virulence, redirecting attention toward non-lytic host-modulatory functions.

   Evidence CRISPR-Cas9 knockout with plaque, egress, and mouse virulence assays in RH strain

   PMID:30211128

   Open questions at the time

   • Negative result does not address chronic infection or dissemination phenotypes
   • Tested only in one strain background
   • No molecular function assigned
2. 2020 Medium

   How GRA28 reaches the host cell was unresolved; identifying TgPPM3C as a phosphatase required for GRA28 export established dephosphorylation as a regulatory checkpoint preceding translocation.

   Evidence Phosphoproteomics and GRA16/GRA28 export assays in ΔTgPPM3C knockout parasites

   PMID:33370417

   Open questions at the time

   • Direct dephosphorylation of GRA28 by TgPPM3C not demonstrated biochemically
   • Which residues are critical for export is undefined
   • Single lab, correlative link between phosphostate and export
3. 2021 High

   The functional output of nuclear GRA28 was unknown; showing it is required for CCL22 induction across human and mouse cells and for in vivo dissemination assigned GRA28 a chemokine-driving, dissemination-promoting role.

   Evidence CRISPR GRA28 deletion, CCL22 ELISA in human/mouse immune and placental cells, in vivo dissemination assay

   PMID:34781732

   Open questions at the time

   • Molecular mechanism linking GRA28 to CCL22 transcription not resolved
   • Direct host DNA/chromatin targets not defined
4. 2022 High

   It was unclear how GRA28 alters host cell behavior; establishing MYR1-dependent nuclear delivery and cooperation with host chromatin remodelers to drive CCR7 upregulation and macrophage hypermigration defined GRA28 as a nuclear reprogramming effector conferring migratory properties.

   Evidence Reciprocal ΔGRA28/ΔMYR1 knockouts, nuclear localization imaging, in vivo competitive adoptive transfer migration assays

   PMID:36309013

   Open questions at the time

   • Identity of the cooperating chromatin remodelers not fully specified
   • Whether GRA28 binds chromatin directly unresolved
5. 2024 High

   The chromatin-level mechanism was undefined; showing GRA28 boosts chromatin accessibility at the Ccr7 locus and enhances GRA15/GRA24/NF-κB-dependent transcription placed GRA28 within a cooperative effector module driving CCR7-mediated chemotaxis and dissemination.

   Evidence Combinatorial GRA knockouts, chromatin accessibility assays, in vivo competitive adoptive transfer in type II strain

   PMID:39207098 PMID:38370679

   Open questions at the time

   • Whether GRA28 acts directly at the locus or via recruited factors unresolved
   • Biochemical partners at the chromatin level not identified
6. 2024 Medium

   GRA28's role in immune-driven cell death was unknown; identifying it among nuclear MYR1-dependent effectors that block IFNγ-driven host cell death, acting by disrupting CBP/p300 acetyltransferase activity and IFNγ-driven transcription, assigned it a direct enzymatic-interference mechanism.

   Evidence Genome-wide loss-of-function screens in IFNγ-primed human cells, combinatorial GRA knockouts, transcription and CBP/p300 acetyltransferase activity assays

   PMID:39292011 PMID:38234811

   Open questions at the time

   • Direct physical interaction between GRA28 and CBP/p300 not established
   • Redundancy with other nuclear effectors complicates isolating GRA28's specific contribution
7. 2026 Medium

   How GRA28 fits into host NF-κB signaling was incompletely mapped; placing it as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-κB (RelB/p52) pathway upstream of TRAF3 depletion and NIK stabilization defined its network-level position.

   Evidence Combinatorial knockout panel, RelB/p52 nuclear accumulation assays, epistasis with MYR1 deletion in type I and type II strains (preprint)

   PMID:41959509

   Open questions at the time

   • Preprint, not yet peer-reviewed
   • GRA28's individual contribution within the additive network not isolated
   • Direct molecular target upstream of TRAF3 depletion unknown

• The direct host molecular target(s) of GRA28 and whether it binds chromatin, CBP/p300, or NF-κB components directly remain undefined.
• No demonstrated direct physical interaction partner for GRA28
• No structural model or defined functional domains
• Mechanism by which GRA28 alters chromatin accessibility is unresolved