GRA20

GRA20 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) but, unlike its Plasmodium PEXEL-motif counterparts, is not exported beyond the PVM into the host cell (PMID:23356236). GRA20 carries an HT/PEXEL-like motif (RxLxD/E) that is cleaved by the Golgi-resident aspartyl protease ASP5; this processing promotes tighter PVM association, whereas cleavage-blocking mutations reduce PVM association without abolishing PV targeting (PMID:26473595, PMID:23356236). CRISPR-Cas9 deletion of GRA20 in the RH strain produces no detectable defect in in vitro growth, egress, or mouse virulence, indicating it is dispensable for these processes in this background (PMID:30211128). Beyond its ASP5-dependent processing and PVM targeting, no molecular effector function for GRA20 has been characterized in the available corpus.

1. 2013 Medium

   It was unknown how GRA20 associates with the PVM and whether, like Plasmodium PEXEL proteins, it is exported into the host cell; localization and mutagenesis established that GRA20 targets the PVM without host export and that HT/PEXEL cleavage strengthens membrane association.

   Evidence HA/Myc tagging with co-localization microscopy and site-directed mutagenesis of the HT/PEXEL motif in T. gondii

   PMID:23356236

   Open questions at the time

   • The identity of the protease responsible for cleavage was not yet defined
   • No molecular partners at the PVM were identified
   • The functional consequence of PVM association for the parasite was not tested
2. 2015 Medium

   It was unclear which protease processes the GRA20 HT/PEXEL motif; deletion of ASP5 showed that this Golgi-resident aspartyl protease is responsible for the cleavage, placing GRA20 processing within the ASP5-dependent maturation pathway.

   Evidence Genetic deletion of ASP5 and biochemical analysis of GRA20 cleavage in T. gondii

   PMID:26473595

   Open questions at the time

   • Downstream functional role of ASP5-mediated GRA20 cleavage beyond membrane association not resolved
   • Single lab, no reciprocal biochemical reconstitution of the cleavage reaction
3. 2018 Medium

   The requirement of GRA20 for parasite fitness was untested; CRISPR-Cas9 deletion in RH established that GRA20 is dispensable for growth, egress, and virulence in this strain, indicating any function is non-essential or redundant in this background.

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

   PMID:30211128

   Open questions at the time

   • Negative result does not exclude functional redundancy with other GRA proteins
   • Not tested in other strains or under stress/immune-competent conditions that might reveal a phenotype
   • No positive molecular function assigned

• The molecular effector function of GRA20 at the PVM and the biological purpose of its ASP5-dependent membrane association remain unknown.
• No binding partners or biochemical activity identified
• No phenotype linking PVM association to parasite biology
• Function in non-RH strains uncharacterized