GRA45

GRA45 is a Toxoplasma gondii dense granule protein that resides within the parasitophorous vacuole (PV) and functions as a component of the effector-translocation machinery required for the parasite to manipulate its host cell (PMID:32075880, PMID:30377279). It physically associates with the MYR1 translocon complex, and its genetic loss abolishes translocation of the dense granule effector GRA16 across the PV membrane and the consequent upregulation of host c-Myc and cyclin E1 (PMID:32075880). GRA45 carries an RRL motif that is cleaved by the Golgi-associated aspartyl protease ASP5, though this proteolytic processing is dispensable for its role in effector translocation (PMID:32075880, PMID:30377279). Consistent with its role in enabling host manipulation, GRA45 acts as an IFN-γ-dependent virulence factor, being required for parasite survival specifically in immune-competent but not Ifngr1-deficient mice (PMID:37269286).

1. 2018 High

   Establishing GRA45 as a genuine ASP5 substrate defined its maturation pathway and confirmed it as a PV-resident dense granule protein, providing the first biochemical foothold on the protein.

   Evidence TAILS N-terminal peptide enrichment and quantitative proteomics comparing wild-type and Δasp5 parasites, with RRL→ARL mutagenesis validation, in Toxoplasma gondii

   PMID:30377279

   Open questions at the time

   • Did not establish the functional role of GRA45 or whether cleavage is required for any function
   • No interacting partners identified
2. 2020 High

   Identifying GRA45 as a MYR1-interacting protein and showing knockouts fail to translocate GRA16 placed GRA45 in the MYR translocon and defined its central function in effector export and host gene modulation.

   Evidence Affinity purification/Co-IP of MYR1 plus GRA45 genetic knockout with GRA16 translocation and host c-Myc/cyclin E1 readouts, in Toxoplasma gondii

   PMID:32075880

   Open questions at the time

   • Whether GRA45 acts directly in the translocation channel or as an accessory/chaperone factor is unresolved
   • The molecular step in translocation that GRA45 enables is not defined
3. 2020 Medium

   Mutating the ASP5 cleavage motif showed that proteolytic maturation is uncoupled from GRA45's translocation function, indicating processing is not a prerequisite for its role in effector export.

   Evidence RRL→ARL site-directed mutagenesis with GRA16 translocation functional assay, in Toxoplasma gondii

   PMID:32075880

   Open questions at the time

   • The functional purpose of ASP5 cleavage of GRA45, if any, remains unknown
   • Single lab, single functional readout
4. 2023 Medium

   An in vivo CRISPR screen with host-genetics epistasis placed GRA45's function in the physiological context of resisting IFN-γ-mediated immune clearance, connecting effector translocation to virulence.

   Evidence In vivo CRISPR screen in C57BL/6 versus Ifngr1-/- mice with comparative enrichment analysis, in Toxoplasma gondii

   PMID:37269286

   Open questions at the time

   • Which specific effectors/host pathways downstream of GRA45 mediate IFN-γ resistance is not defined
   • Single method, single lab

• It remains unknown whether GRA45 forms a structural part of the translocation channel or acts as a chaperone/accessory factor, and what the full repertoire of GRA45-dependent effectors and host pathways is.
• No structural model of GRA45 within the MYR complex
• Full effector-dependency profile beyond GRA16 undefined
• Molecular mechanism linking GRA45 to IFN-γ resistance unresolved