GRA14

GRA14 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole, where it adopts a transmembrane topology at the PV membrane (PVM) and intravacuolar network (IVN) with its C-terminus exposed to the host cytoplasm and its N-terminus in the vacuolar lumen (PMID:18765740). Through late-domain-like motifs homologous to those of HIV-1 Gag, GRA14 recruits host ESCRT machinery to the PVM—its motif-containing region can functionally substitute for the HIV-1 Gag late domain in ESCRT-dependent virus-like particle budding—and it interacts with host ESCRT components at the vacuole (PMID:34898650). This ESCRT recruitment drives PVM invaginations and the vesicular uptake of host cytosolic proteins and organelles into the PV, an activity that GRA14 shares with GRA64 and that is more severely impaired in a double knockout (PMID:34898650, PMID:36718630). The resulting ingestion pathway contributes to nutrient scavenging, as Δgra14 parasites show reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation (PMID:40172222). GRA14 is also partially required for activation of host NF-κB signaling, promoting RelA nuclear translocation and IL-6 production in infected macrophages (PMID:32849602), and is required in vivo for chronic-stage cyst development (PMID:31266861). GRA14 can be transferred between vacuoles during coinfection, indicating mobility between PVs (PMID:18765740).

1. 2008 High

   Before its topology was defined, it was unknown how a dense granule protein could act at the host-parasite interface; this work established GRA14 as a PVM/IVN transmembrane protein with its C-terminus facing the host cytoplasm, positioning it to engage host machinery.

   Evidence Subcellular fractionation, immunofluorescence, and protease protection topology assays with a GRA14 knockout strain

   PMID:18765740

   Open questions at the time

   • Host-facing binding partners not identified at this stage
   • Functional consequence of the topology unresolved
2. 2008 Medium

   It was unclear whether dense granule proteins are static within their vacuole; coinfection transfer of GRA14 from wild-type to knockout vacuoles demonstrated intervacuolar mobility.

   Evidence Coinfection of GRA14 knockout and wild-type parasites with immunofluorescence detection of transferred GRA14

   PMID:18765740

   Open questions at the time

   • Mechanism and route of transfer between PVs unknown
   • Functional relevance of transfer not established
3. 2018 Medium

   To test whether GRA14 is required for acute infection, its deletion in the virulent type I RH strain was assessed; loss had no effect on in vitro growth, egress, or acute virulence, showing GRA14 is dispensable for these processes in this strain.

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

   PMID:30211128

   Open questions at the time

   • Negative finding restricted to RH acute infection
   • Chronic-stage role not addressed in this strain
4. 2019 High

   The in vivo role of GRA14 was unresolved; deletion in the type II Prugniaud strain revealed a specific defect in chronic-stage cyst development without affecting in vitro growth or differentiation, establishing GRA14 in chronic infection.

   Evidence GRA14 knockout in Prugniaud with in vivo mouse infection and in vitro cyst formation assays

   PMID:31266861

   Open questions at the time

   • Molecular basis linking GRA14 to cyst persistence unresolved
   • Relationship to its PVM/ingestion functions not established
5. 2020 Medium

   Whether GRA14 modulates host innate signaling was unknown; its deletion in Pru reduced RelA nuclear translocation, IL-6 production, and NF-κB-driven transcription, implicating GRA14 partially in host NF-κB activation.

   Evidence NF-κB luciferase reporter, GRA14 knockout in Pru, RelA nuclear translocation assay, IL-6 ELISA, and RNA-seq in macrophages

   PMID:32849602

   Open questions at the time

   • Effect is partial and mechanism of NF-κB activation unresolved
   • Direct versus indirect involvement not distinguished
6. 2021 High

   How the parasite co-opts host membrane-remodeling machinery was unknown; GRA14 was shown to recruit host ESCRT via HIV-1 Gag-like late-domain motifs and to be required for host cytosolic protein ingestion, defining a molecular mechanism for host-material uptake.

   Evidence HIV-1 VLP release functional substitution assay, proximity/co-IP with host ESCRT components, and GRA14 knockout ingestion assay

   PMID:34898650

   Open questions at the time

   • Precise ESCRT subunit engaged by the late-domain motif not fully mapped
   • Stoichiometry with other dense granule proteins unresolved
7. 2023 High

   It was unclear whether ESCRT recruitment drives PVM membrane deformation and organelle uptake; GRA14 (with GRA64) was shown required for ESCRT-driven PVM invaginations and scavenging of host organelles, with a stronger double-knockout defect.

   Evidence Dominant-negative CHMP4B and Vps4A constructs, GRA14/GRA64 single and double knockouts, and microscopy of PVM invaginations and intra-PV organelles

   PMID:36718630

   Open questions at the time

   • Functional division of labor between GRA14 and GRA64 not resolved
   • Selectivity of organelle capture unknown
8. 2025 Medium

   Whether the GRA14-initiated ingestion pathway feeds parasite metabolism was open; Δgra14 parasites showed reduced glycolytic intermediates and amino acids and a growth defect under amino acid limitation, linking GRA14-driven ESCRT recruitment to host nutrient scavenging.

   Evidence Genome-wide CRISPR fitness screen, bulk metabolomics, and growth assays in amino acid-depleted media in WT and Δgra14/Δcpl/Δcrt mutants

   PMID:40172222

   Open questions at the time

   • Evidence for GRA14's nutrient role is partly inferred from downstream mutants
   • Specific nutrients delivered via the pathway not directly demonstrated

• How GRA14's transmembrane topology, ESCRT-recruiting ingestion function, NF-κB modulation, and requirement for chronic cyst development are mechanistically integrated remains unresolved.
• No mechanism links GRA14-driven ingestion to chronic cyst persistence
• The host receptor/ESCRT subunit specificity of the late-domain motif is not fully defined
• Direct role in NF-κB signaling versus indirect consequence is unresolved