GRA35

GRA35 is a Toxoplasma gondii dense granule protein that acts as a multifunctional effector at the parasite–host interface, engaging host cell death and immune restriction pathways from the parasitophorous vacuole membrane (PVM) (PMID:30622189, PMID:38376248). Its correct trafficking to the PVM requires GRA42 and GRA43, and loss of either retains GRA35 within the parasitophorous vacuole; all three proteins function in a common pathway required to induce Lewis rat macrophage pyroptosis (PMID:30622189). At the PVM, GRA35 directly binds the host E3 ubiquitin ligase ITCH, an interaction required for NLRP1 inflammasome activation and pyroptosis consistent with a proteasome-dependent NLRP1 functional-degradation model (PMID:38376248). The same GRA35–ITCH interaction restricts parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM (PMID:38376248). In ME49 chronic brain infection, GRA35 additionally promotes neuronal apoptosis by binding RTN1-c to engage the mitochondrial apoptosis pathway and by inducing PERK-mediated ER stress (PMID:41940502). Beyond its role in pyroptosis, GRA35 (together with GRA42 and GRA43) contributes to in vivo fitness and chronic cyst establishment in rats independently of inflammasome activation (PMID:30622189).

1. 2019 High

   Establishing which parasite factors drive host macrophage pyroptosis, a forward-genetic screen defined GRA35, GRA42, and GRA43 as components of a single pathway required for cell death and replication control.

   Evidence Chemical mutagenesis screen with whole-genome sequencing, targeted single/double/triple deletions, and macrophage infection assays in Lewis rats

   PMID:30622189

   Open questions at the time

   • Molecular event triggering pyroptosis downstream of the three GRAs was not defined
   • Direct host binding partner of GRA35 not yet identified
2. 2019 High

   To place the three GRAs in a spatial hierarchy, localization studies showed GRA42 and GRA43 are required to deliver GRA35 to the PVM, establishing them as trafficking determinants for GRA35 function.

   Evidence Immunofluorescence localization of GRA35 in Δgra42 and Δgra43 parasites

   PMID:30622189

   Open questions at the time

   • Mechanism by which GRA42/GRA43 mediate PVM delivery unknown
   • Whether GRA42/GRA43 act during translocation or membrane insertion not resolved
3. 2019 Medium

   Testing whether the GRAs matter beyond acute cell death, in vivo infection revealed they support chronic cyst establishment independently of inflammasome/pyroptosis activation.

   Evidence Gene deletion and cyst burden quantification in F344 rats

   PMID:30622189

   Open questions at the time

   • Molecular basis of the pyroptosis-independent fitness role unknown
   • Host pathway underlying reduced cyst numbers not identified
4. 2024 High

   Identifying the host receptor for GRA35's pyroptotic activity, an unbiased CRISPR screen and interaction studies showed GRA35 directly binds the E3 ubiquitin ligase ITCH to activate the NLRP1 inflammasome via a proteasome-dependent functional-degradation mechanism.

   Evidence Genome-wide CRISPR screen, immunofluorescence, Co-IP/pulldown, ITCH knockout, and proteasome inhibitor experiments in Lewis rat cells

   PMID:38376248 PMID:38168400

   Open questions at the time

   • No evidence that ITCH directly ubiquitinates rat NLRP1
   • Structural basis of the GRA35–ITCH interaction unresolved
   • How ITCH engagement couples to NLRP1 degradation not fully mechanistically defined
5. 2024 Medium

   Extending the GRA35–ITCH axis to cell-autonomous immunity, the interaction was shown to reduce parasite fitness in IFNγ-activated human fibroblasts by recruiting ubiquitin and the restriction factor RNF213 to the PVM.

   Evidence IFNγ-activated human fibroblast infection assays, ITCH knockout, and RNF213 localization assays

   PMID:38376248

   Open questions at the time

   • Direct causal link between RNF213 recruitment and restriction not fully established
   • Whether GRA35 confers advantage or liability across cell types not reconciled
6. 2026 Medium

   Probing GRA35's role in chronic CNS infection, GRA35 was shown to bind RTN1-c and drive neuronal apoptosis via both the mitochondrial pathway and PERK-mediated ER stress.

   Evidence GRA35 knockout in ME49, GST pulldown (GRA35–RTN1-c), immunofluorescence, flow cytometry, and CCK8 assays in brain neurons

   PMID:41940502

   Open questions at the time

   • GST pulldown interaction not validated by reciprocal or in vivo methods
   • How PVM-localized GRA35 accesses neuronal RTN1-c and ER machinery not defined
   • Relationship between apoptosis induction and inflammasome/ITCH functions unclear

• How GRA35's distinct host-directed activities—NLRP1/pyroptosis, ubiquitin/RNF213 restriction, and RTN1-c/PERK-driven neuronal apoptosis—are integrated at the domain level and coordinated across host cell types and infection stages remains unknown.
• No structural or domain-mapping data linking the multiple binding activities of GRA35
• No unified model reconciling parasite-protective versus parasite-restrictive outcomes