Toxoplasma–host interactions, gene by gene

Toxoplasma gondii infects roughly one third of people worldwide and nearly every warm-blooded animal. Most infections are silent, yet the parasite can cause devastating disease in the developing fetus, in immunocompromised people and in the eye. We want to understand, gene by gene, how Toxoplasma establishes itself inside a host cell and spreads through the body, and why some hosts and some parasite strains produce very different outcomes.

Toxoplasma CRISPR screens

Genome-wide CRISPR/Cas9 loss-of-function screens in Toxoplasma let us ask which of the parasite’s roughly 8,000 genes matter in a given setting: in human fibroblasts, in naive or interferon-γ-activated mouse and human macrophages, in lipid-rich versus lipid-poor medium, in parasites that already lack a key gene (synthetic lethality), and directly in mice, where fitness in the peritoneum is compared with fitness in distant organs.

The screens tell us which parasite genes matter. The follow-up work, in cells, in mice and rats, and with purified proteins, tells us how.

Toxoplasma effectors and dissemination

To cause systemic infection, Toxoplasma must cross biological barriers, disseminate to distant organs including the brain, and avoid innate immune clearance. It does so largely through secreted Toxoplasma effectors: the rhoptry proteins (ROPs) injected during invasion and the dense granule proteins (GRAs) released into and beyond the parasitophorous vacuole. Using CRISPR screens performed in vivo we map the parasite genes required for fitness in the peritoneum and in organs.

One of them, TgWIP, is secreted into dendritic cells, where it reprograms actin dynamics and motility through the phosphatase SHP2 and the WAVE and Arp2/3 complexes, work done together with Antonio Barragan’s group at Stockholm University. Related effectors, including GRA28, give infected macrophages dendritic-cell-like hypermigration.

Immune evasion: surviving interferon-γ

Interferon-γ is the central mediator of resistance to Toxoplasma, and macrophages are frontline defenders. A genome-wide screen in naive and IFNγ-activated mouse macrophages identified about 500 parasite genes that affect fitness. GRA45, for example, keeps other effectors from aggregating before secretion; parasites lacking it are hypersensitive to IFNγ and attenuated in mice.

Human cells restrict the parasite differently. ROP5 and ROP18, essential for virulence in mice, are dispensable in human cells, so we screen directly in IFNγ-stimulated human fibroblasts and macrophages. These screens identified the GRA70 complex, a set of dense granule proteins the parasite needs specifically in IFNγ-stimulated human cells, and showed that the host E3 ubiquitin ligase ITCH links the effector GRA35 to NLRP1 inflammasome activation. We also study how human immune cells first recognize the parasite, including the early IFNγ response mounted by Vγ9Vδ2 T cells.

Life inside the parasitophorous vacuole

The parasite replicates inside a parasitophorous vacuole that separates it from the host cytosol, so everything it eats must cross the vacuole membrane. Dense granule proteins GRA17 and GRA23 form the pores that let small molecules diffuse in; GRA47 and GRA72 tune that permeability, and synthetic-lethality screens in parasites lacking GRA17 reveal the back-up routes. More recently we found GRA38, a regulator of parasite lipid homeostasis, and TgSEC14-LTP1, a SEC14-like lipid-transfer protein secreted into the vacuole.

Host and parasite genetics of toxoplasmosis

Toxoplasma strains differ enormously in virulence. Profiling macrophages infected with 29 diverse strains showed how strain-specific effectors such as ROP16 and GRA15 steer STAT3/6 and NF-κB signaling, and with it inflammation. On the host side, macrophages from resistant rat strains die rapidly through the NLRP1 inflammasome upon infection, denying the parasite a niche, whereas mouse macrophages activate inflammasomes without dying.

The same questions reach beyond the laboratory mouse: the strains behind ocular disease in South America, the proteins that let oocysts survive in the environment, and, in a collaboration led by Karen Shapiro’s lab at UC Davis that grew out of our peptide-based serotyping work, the Toxoplasma strains that kill southern sea otters off the California coast.