GRA42

GRA42 is a Toxoplasma gondii parasitophorous vacuole (PV)-localized dense granule protein that acts as an effector controlling the correct trafficking of other GRA proteins and the activation of host cell death (PMID:30622189). GRA42 is required for the proper localization of GRA35 and additional GRAs to the parasitophorous vacuole membrane (PVM); in Δgra42 parasites these GRAs are retained inside the PV rather than reaching the PVM (PMID:30622189). GRA42 functions in a common pathway with GRA35 and GRA43, since single, double, and triple deletions of these genes produce equivalent loss of Lewis rat macrophage pyroptosis (PMID:30622189), and deletion of GRA42 greatly reduces macrophage death while enhancing parasite replication (PMID:30622189); an unbiased genome-wide CRISPR screen independently confirmed GRA35, GRA42, and GRA43 as the effectors mediating this cell death (PMID:38376248). Beyond its role in pyroptosis, GRA42 is also required for parasite in vivo fitness in susceptible F344 rats, as Δgra42 parasites form fewer brain cysts, a role independent of macrophage pyroptosis (PMID:30622189). The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking have not been characterized in the available corpus.

1. 2019 High

   Establishing that a defined parasite effector controls host macrophage cell death, GRA42 was identified as a PV-localized dense granule protein individually required for Lewis rat macrophage pyroptosis.

   Evidence Chemical mutagenesis screen with whole-genome sequencing and targeted Δgra42 deletion, assayed by macrophage cell death and parasite replication

   PMID:30622189

   Open questions at the time

   • molecular activity of GRA42 not defined
   • direct host target linking GRA42 to pyroptosis not identified
2. 2019 High

   To explain how GRA42 acts, it was shown to be required for trafficking of GRA35 and other GRAs from the PV lumen to the PVM, positioning GRA42 as a mediator of effector localization rather than a direct death inducer.

   Evidence Immunofluorescence comparison of GRA35 localization in wild-type versus Δgra42 parasites

   PMID:30622189

   Open questions at the time

   • mechanism by which GRA42 directs GRA trafficking to the PVM unknown
   • whether GRA42 physically interacts with the trafficked GRAs not shown
3. 2019 High

   Genetic epistasis placed GRA35, GRA42, and GRA43 in a single pathway for pyroptosis induction, defining their functional relationship.

   Evidence Comparison of single, double, and triple deletion parasites with quantitative macrophage viability readouts

   PMID:30622189

   Open questions at the time

   • molecular hierarchy among the three GRAs within the pathway not resolved
   • host inflammasome components engaged not defined in this evidence
4. 2019 Medium

   A distinct in vivo role emerged when Δgra42 (and Δgra35, Δgra43) parasites showed reduced brain cyst burden in susceptible F344 rats, establishing a pyroptosis-independent fitness function.

   Evidence In vivo F344 rat infection with knockout parasites and brain cyst quantification

   PMID:30622189

   Open questions at the time

   • no orthogonal mechanism identified for the pyroptosis-independent fitness role
   • single-lab in vivo finding
5. 2024 High

   An unbiased genome-wide screen validated the earlier candidate-based findings, confirming GRA35, GRA42, and GRA43 as the parasite effectors driving macrophage pyroptosis.

   Evidence Genome-wide CRISPR screen in Lewis rat macrophages infected with Toxoplasma gondii

   PMID:38376248

   Open questions at the time

   • screen confirms requirement but does not define GRA42 biochemical function
   • does not resolve the trafficking mechanism

• The molecular activity of GRA42 and the mechanism by which it directs GRA trafficking to the PVM and enables NLRP1-dependent pyroptosis remain unresolved.
• no biochemical activity assigned to GRA42
• no structural model or interaction partner identified
• mechanistic basis of the pyroptosis-independent in vivo fitness role unknown