Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon

Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)-and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro. Acute virulence of the Pru Delta gra2, Pru Delta gra3, and Pru Delta gra4 mutants was reduced but not abolished. In contrast, the Pru Delta gra12 mutant was avirulent in mice and Pru Delta gra12 parasites failed to establish a chronic infection. High virulence type I strain RH Delta gra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-gamma)-activated macrophages, type I RH Delta gra12 and type II Pru Delta gra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RH Delta ku80 and type II Pru Delta ku80 strains, respectively. Despite this resistance, Delta gra12 PVs ultimately succumbed to IFN-gamma-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-gamma and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection. IMPORTANCE Toxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo. A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-gamma). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.