mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

Abstract

AbstractGuanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host’s intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii. Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.Significance StatementWe have previously shown that mGBP2 is an essential IFNγ induced protein for defense against Toxoplasma gondii infection. mGBP2 is rapidly recruited to the parasitophorous vacuole (PV) of intracellular T. gondii and is required for the rupture and/or permeabilization of the PV membrane. Subsequently mGBP2 attacks the parasite membrane. Up to now, no data about other mGBP2 interaction partners and their molecular requirement for T. gondii control has been made available.Here, we have identified Gal9 and Ckap4 as interacting proteins of mGBP2. Gal9 and Ckap4 are essential in the mGBP2 mediated attack of the T. gondii PV. Both proteins localize at the T. gondii PV in close association with mGBP2, as shown by state-of-the-art super resolution microscopy. In addition, T. gondii growth cannot be controlled anymore in Gal9 and Ckap4 deficient cell lines, comparable to mGBP2 deficient cells,.Thus, we have identified novel effector proteins in the intricate cell autonomous immune machinery against intracellular pathogens.