APEX2 proximity labeling maps the mGBP2-proximal proteome at the Toxoplasma gondii parasitophorous vacuole

Abstract

Interferon-inducible guanylate binding proteins (GBPs) are large GTPases crucial for the host immune response against Toxoplasma gondii, a globally prevalent intracellular apicomplexan parasite. Upon infection, T. gondii forms a parasitophorous vacuole (PV) that protects the parasite from host intracellular defense mechanisms. Murine GBPs (mGBPs) can recognize these PVs and assemble into supramolecular homo- and heterocomplexes, which is an essential step for PV membrane disruption, ultimately enabling cell-autonomous immune control of vacuole-resident pathogens. Our previous work has shown that mGBP2 is an important factor in the immune control of T. gondii. To further elucidate the functions of mGBP2, we employed an APEX2-based proximity labeling strategy to biotinylate proteins in the vicinity of mGBP2 during T. gondii infection. Mass spectrometry analysis of the biotinylated proteins revealed several novel PV-associated proteins such as Cyclooxygenase 2 (COX2/ Ptgs2), Caspase 12 (Cas12), Annexin 11 (Anxa11), Calnexin (Canx), Galectin 3 (Gal3), and Transgelin (Tagln) and valosin-containing protein (VCP) that co-recruit with mGBP2 at T. gondii PVs. In addition, we investigated their regulation by IFN-{gamma} and determined that inhibition of COX2 severely impairs T. gondii growth, highlighting a critical role for COX2 for parasite survival.