Identification and characterisation of Toxoplasma gondii effectors mediating parasite virulence in human cells

Abstract

Toxoplasma gondii is an intracellular eukaryotic parasite that chronically infects approximately one third of the global human population. To establish an infection, Toxoplasma must initially avoid clearance by disarming the human immune system. To do this, Toxoplasma secretes over two hundred effector proteins, which collectively reshape the intracellular environment and resist innate immune responses. Previous efforts to characterise Toxoplasma secreted effectors largely centered on their role in the murine host. However, innate immune control of Toxoplasma has significantly diverged between humans and mice, therefore effector functions are not always conserved between the two hosts. Relatively little is known about which secreted effectors are required for Toxoplasma to evade the human immune response. In this work I used targeted CRISPR screening of Toxoplasma secreted effectors to identify which are required for survival in interferon-gamma-activated human cells. CRISPR screens were carried out using two Toxoplasma strains that differ in virulence in mice. From these screens I found that a complex of secreted dense granule proteins (GRA57, GRA70, and GRA71) resists interferon-gamma-induced parasite clearance in human fibroblasts. This complex was conversely dispensable for parasite survival in mouse fibroblasts, suggesting it may have species-specific functions. Additionally, I found that multiple components of the Toxoplasma effector export machinery are required for immune resistance in human and mouse fibroblasts. I next investigated the function of the GRA57, GRA70, and GRA71 complex, and ruled out roles in effector export, transcriptional modulation, tryptophan acquisition or formation of the parasite vacuole. I instead found that GRA57, GRA70, and GRA71 knockout parasite vacuoles are less ubiquitinated by the host cell, but this is uncoupled from their function in immune resistance. Together these results advance our previously limited understanding of which Toxoplasma effector proteins are required for the parasite to establish human infections.