GRA43

GRA43 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole and functions as a trafficking factor required for delivering GRA35 and other dense granule proteins to the parasitophorous vacuole membrane (PVM) (PMID:30622189). Loss of GRA43 causes GRA35 and other GRAs to be retained inside the vacuole rather than reaching the PVM (PMID:30622189), and genetic epistasis places GRA43 in a common pathway with GRA35 and GRA42 (PMID:30622189). Through this PVM-organizing role GRA43 acts upstream of GRA35's engagement of the host ITCH E3 ubiquitin ligase at the PVM to drive pyroptotic macrophage death in Lewis rats, such that GRA43 deletion strongly reduces cell death and enhances parasite replication (PMID:30622189, PMID:38376248). GRA43 also contributes to parasite in vivo fitness in susceptible F344 rats independently of its inflammasome role (PMID:30622189).

1. 2019 High

   Establishing whether a specific parasite effector controls host macrophage death, this work identified GRA43 as a PV-localized dense granule protein individually required for Lewis rat macrophage pyroptosis and placed it in a shared pathway with GRA35 and GRA42.

   Evidence Chemical mutagenesis screen with whole-genome sequencing, CRISPR knockout, viability assays, and single/double/triple deletion epistasis

   PMID:30622189

   Open questions at the time

   • Molecular mechanism by which GRA43 promotes trafficking not defined
   • No direct binding partners of GRA43 itself identified
2. 2019 High

   To explain how GRA43 enables downstream pyroptosis, deletion showed GRA43 is needed to traffic GRA35 and other dense granule proteins to the PVM rather than retaining them in the vacuole, defining its role as a PVM-targeting factor.

   Evidence Δgra42 and Δgra43 knockout parasites with fluorescence localization of GRA35 and other GRAs

   PMID:30622189

   Open questions at the time

   • Whether GRA43 physically escorts cargo or organizes a translocation machinery is unknown
   • Full set of GRA43-dependent cargo not enumerated
3. 2019 Medium

   To separate GRA43's inflammasome role from other functions, in vivo infection revealed GRA43 is required for parasite fitness in susceptible F344 rats independently of NLRP1/pyroptosis activation.

   Evidence In vivo infection of F344 rats with Δgra43 parasites and brain cyst counting

   PMID:30622189

   Open questions at the time

   • Single lab and single method for the F344 fitness phenotype
   • Mechanistic basis of the fitness defect not resolved
4. 2021 Medium

   Probing how effector secretion converts to macrophage death, GRA43 secretion was linked to ROS induction that damages PVM and mitochondrial membranes, depleting SOD2 and driving pyroptosis in iNOS-knockout rat macrophages.

   Evidence iNOS-knockout rat macrophage infection, ROS measurement, and organelle membrane integrity assays

   PMID:34765911

   Open questions at the time

   • ROS attribution relies on correlation with GRA43 secretion rather than a direct Δgra43 control in this system
   • How ROS mechanistically connects to the GRA35/GRA42 pathway is unclear
5. 2023 Low

   Extending GRA43's immune impact beyond pyroptosis, genetic analysis indicated GRA43 represses the CD8 T cell IFNγ differentiation response, implicating PVM-associated GRAs in modulating CD8 T cell activation.

   Evidence QTL mapping with F1 progeny and co-culture of Δgra43-infected macrophages with antigen-specific CD8 T cells with IFNγ readout

   PMID:37287466

   Open questions at the time

   • Observed as a secondary note rather than primary experimental focus; single lab and single method
   • Direct mechanism linking GRA43-dependent PVM localization to CD8 T cell suppression not defined
6. 2024 High

   Independently confirming and ordering the pathway, a genome-wide CRISPR screen validated GRA43 (with GRA35 and GRA42) as a cell-death effector acting upstream of GRA35's interaction with host ITCH at the PVM.

   Evidence Genome-wide CRISPR screen in Lewis rat macrophages

   PMID:38376248

   Open questions at the time

   • Direct biochemical interaction between GRA43 and any host or parasite factor still not shown
   • Structural basis of GRA43 trafficking function unresolved

• The molecular mechanism by which GRA43 directs dense granule proteins to the PVM — its direct binding partners, cargo recognition, and biochemical activity — remains uncharacterized in the available corpus.
• No direct GRA43 interactome
• No structural model
• Mechanism of cargo selection and PVM targeting unknown