GRA64

GRA64 is a Toxoplasma gondii transmembrane dense granule protein that functions at the host-parasite interface to co-opt host membrane-remodeling machinery (PMID:35730903, PMID:36718630). It localizes to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite vacuoles, with a domain partially exposed to the host cytoplasm where it engages host endosomal sorting complexes required for transport (ESCRT) (PMID:35730903). Through this exposed region, GRA64 cooperates with GRA14 to recruit host ESCRT-III (CHMP4B) to the PVM, driving ESCRT-dependent PVM invaginations and the scavenging of host organelles into the parasitophorous vacuole; loss of GRA64 reduces these invaginations and organelle accumulation, with the strongest defect in the GRA14/GRA64 double knockout (PMID:36718630). During chronic infection, GRA64 disruption produces enlarged vesicular structures beneath the cyst membrane, implicating it in organizing intracystic vesicle formation (PMID:35730903).

1. 2022 Medium

   Establishing where GRA64 acts and how it is oriented was the first step in defining its role at the host-parasite interface; localizing it to the PVM with host-cytoplasm exposure positioned it to interact with host factors.

   Evidence Immunofluorescence, ultrastructural analysis, and subcellular fractionation of tachyzoite and bradyzoite vacuoles in T. gondii ME49

   PMID:35730903

   Open questions at the time

   • Precise topology and which residues face the host cytoplasm not mapped
   • Membrane insertion/trafficking mechanism to the PVM not defined
2. 2022 Medium

   Identifying host binding partners answered what GRA64's cytoplasm-exposed domain does; two orthogonal methods showed it engages host ESCRT components at the PVM.

   Evidence Co-immunoprecipitation and proximity-based biotinylation (BioID) in T. gondii ME49

   PMID:35730903

   Open questions at the time

   • Direct versus indirect binding to specific ESCRT subunits not resolved
   • Interaction interface not defined
3. 2022 Medium

   Genetic loss-of-function connected GRA64 to a chronic-stage structural phenotype, showing it is required for proper intracystic vesicle organization during bradyzoite/cyst development.

   Evidence Δgra64 knockout with electron tomography of tissue cysts in T. gondii ME49

   PMID:35730903

   Open questions at the time

   • Causal link between altered vesicles and parasite fitness/persistence not established
   • Whether the phenotype reflects ESCRT mis-recruitment not directly tested
4. 2023 High

   Reciprocal genetic epistasis with GRA14 and dominant-negative ESCRT perturbation established GRA64's mechanistic role: it drives ESCRT-III-dependent PVM invagination and scavenging of host organelles into the vacuole.

   Evidence Single and double knockouts (ΔGRA64, ΔGRA14, double KO) with dominant-negative CHMP4B and Vps4A expression, fluorescence and electron microscopy in T. gondii

   PMID:36718630

   Open questions at the time

   • Molecular basis of cooperativity between GRA64 and GRA14 not defined
   • Functional consequence of host organelle scavenging for parasite metabolism not established
   • Order of ESCRT subunit recruitment relative to GRA64 not resolved

• How GRA64 physically nucleates ESCRT-III assembly and whether its chronic-stage vesicle role is mechanistically identical to its tachyzoite scavenging function remain open.
• No structural model of the GRA64-ESCRT interaction
• Direct binding partner among ESCRT subunits not identified
• Contribution to virulence or persistence in vivo not quantified