Abstract
Toxoplasma gondii is an incredibly successful parasite due, in part, to its ability to persist within cells for the life of the host. Remarkably, at least 350 host species of T. gondii have been described to date and it is estimated that 30% of the global human population is chronically infected. The AIDS crisis in the 1980s revealed the prevalence of chronic infection, as patients presented with reactivated chronic toxoplasmosis, underscoring the importance of an intact immune system for parasite control. In the last 40 years, there has been tremendous progress toward understanding the host-T. gondii interaction using rodent models, human cell experimental systems and clinical data. However, there are still major holes in our understanding of innate immune response to T. gondii infection, including the mechanisms of cell-intrinsic immunity to T. gondii as well as the fate of host cells during parasite infection. In this thesis, we present two major studies demonstrating that inducible nitric oxide synthase (iNOS) is necessary for chromosome 3 guanylate binding protein (GBP)-mediated parasite clearance through collapsing intravacuolar network (IVN) space and inhibiting egressing of GBP2-targeted parasite; and absent in melanoma 2 (AIM2) inflammasome detects host derived DNA during T. gondii infection to activate cell death.
Using automated spatially targeted optical micro proteomics (autoSTOMP) we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.
Mouse bone marrow derived monocytes and dendritic cells (BM-MoDCs) undergo inflammasome activation, IL-1β release, and lytic cell death in response to T. gondii infection during IFNγ-mediated parasite clearance. The IL-1β release depends on both IFNγ and TLR2 signaling in response to T. gondii infection. AIM2 inflammasome is the main sensor for the parasite and to a lesser extent, NLRP3 also participates in the inflammasome activation which leads to caspase-1, GSDMD cleavage, and mature IL-1β release. Using UV-irradiated and heat-killed parasite we find that active parasite invasion is required for the inflammasome activation, however, rescuing parasite killing using iNOS-deficient BM-MoDCs does not affect inflammasome activation, suggesting DNA source other than parasite is detected by AIM2 inflammasome. Depleting host mitochondrial DNA (mtDNA) impairs inflammasome activation to the level of Aim-/- cells and mtDNA is detected in the cytosol. Our data indicate that host mtDNA release induced by T. gondii infection activates AIM2 inflammasome in IFNγ and TLR2 activated macrophage.
In conclusion, this thesis presents complex cooperation between different IFNγ-mediated parasite clearance pathways and the interruption of host cell homeostasis which ultimately leads to inflammasome activation and host cell death.
