p97/VCP targets Toxoplasma gondii vacuoles for parasite restriction in interferon-stimulated human cells

Infection with the parasite Toxoplasma gondii leads to production of interferon gamma (IFNγ) that stimulates cells to upregulate defense proteins targeting the parasite for cell intrinsic elimination or growth restriction. Various host defense mechanisms operate at the parasitophorous vacuole (PV) in different human cell types leading to PV disruption, acidification, or membrane envelopment. Ubiquitin and p62 are players in all human host control mechanisms of Toxoplasma, but other unifying proteins have not been identified. Here, we show that p97/valosin-containing protein (VCP), as well as its associated proteins ANKRD13A and UBXD1 control Toxoplasma infection while recruited to the PV in IFNγ-stimulated endothelial cells. Convergent deposition of ANKRD13A, p97/VCP, and UBXD1 onto the same vacuole is dependent on vacuolar ubiquitination and observed within 2 h post-infection. ANKRD13A, p97/VCP, and UBXD1 all drive the acidification mechanism of the vacuole, which is the IFNγ-dependent control pathway of Toxoplasma in endothelial cells. We assessed p97/VCP in Toxoplasma control in various human cells and demonstrate that p97/VCP is a universal IFNγ-dependent host restriction factor targeting the Toxoplasma PV in epithelial (HeLa) and endothelial cells (human umbilical vein endothelial cells), fibroblasts (human foreskin fibroblast), and macrophages (THP1).