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.