Toxoplasma gondii is a global health hazard, estimated to infect 30-50% of the world population. Toxoplasma has a complicated life cycle: it can be found either as sporozoites in oocysts in the intestines of felids - their definitive hosts – or as tachyzoites (the actively multiplying, disease-causing stage) or bradyzoites (the slowly multiplying stage) enclosed in tissue cysts of intermediate hosts such as mice and humans, in eye, muscle and neural tissue. Humans typically become infected via ingestion of oocysts in contaminated food or water or undercooked meat with parasite cysts. Shortly after ingestion, oocysts hatch and cyst walls are dissolved and motile tachyzoites, capable of establishing an intracellular niche, multiply and invade host tissues.
Intracellular T. gondii resides and replicates inside a membrane-bound organelle, the parasitophorous vacuole (PV), where it avoids detection and elimination; its progeny invades neighbouring cells, propagating the infection. In the absence of an adequate immune response, uncontrolled tachyzoite replication can have devastating effects (encephalitis, blindness, even death), particularly during pregnancy and in immunocompromised individuals. Some aggressive T. gondii strains can even kill people with fully functional immune systems.
Although concentrated in Central and South America, the toxoplasmosis burden in Europe is high: it causes ~20% of foodborne diseases, affecting > 2 million Europeans/year (WHO 2015 estimates). Drugs that treat toxoplasmosis fail to eradicate cysts and often cause drug resistance. Yet, most Toxoplasma infections are silent: a vigorous cell-autonomous (innate) immunity (CAI) response orchestrated by interferon gamma (IFNγ) limits parasite dissemination in the host and drives (asymptomatic) latency.
My focus was to gain a greater understanding of events taking place in IFNγ-activated cells shortly after infection, which are determinant for infection outcome. Mice are primarily reliant on CAI effectors virtually absent in humans, the immunity-related GTPases (IRGs), whereas humans are thought to rely mainly on guanylate-binding proteins (GBPs). In mouse cells, PV membrane (PVM) attack by IRGs/GBPs results in parasite eviction from its intracellular niche into the host cytoplasm. Parasite death is swiftly followed by regulated necrosis of the host cell, a pro-inflammatory type of cell death. The innate immune system deploys pattern-recognition receptors (PRRs) that detect pathogens directly, by recognizing pathogen-associated molecular patterns (PAMPs), or indirectly, by recognizing damage-associated molecular patterns (DAMPs) that result from cell death or tissue damage. Upon detection of microbial presence or damage to cellular structures PRRs trigger immune responses that may be beneficial and limit infection or cause immunopathology. The latter can lead to inflammatory disorders, a major clinical burden worldwide, and even host death.
Preciously little was known about how Toxoplasma is detected by host PRRs or how parasite sensing drives host cell death (Figure 1). I had 2 objectives: 1) to identify the PRR responsible for Toxoplasma sensing in the cytoplasm and 2) to identify the pathway(s) by which the host cell undergoes regulated necrosis.
My work has provided much needed elucidation. Using genetic screens and cell biology tools to pinpoint the PRR/necrosis signalling genes involved, I uncovered a role for galectin-8 (and galectin-9) in Toxoplasma sensing (Figure 2), likely via recruitment of the selective autophagy machinery. I excluded a role for cytosolic PRRs of the RLR, TLR and DNA sensor families and the MyD88-TRIF-IPS-1/MAVS-STING signaling axes. Uncovering any additional sensor(s) of Toxoplasma is now simplified.
My necrosis screen yielded 3 surprising findings: 1) RIPK3-MLKL-RIPK1-Caspase 8 necrosome assembly is not required for fibroblast necrosis, excluding a requirement for canonical necroptosis; 2) my data hint at a role for inflammasomes, not typically associated with fibroblast necrosis; 3) by probing a role for mitochondrial and lysosomal cell death, I discovered novel parasite invasion inhibitors. My work has allowed a more accurate picture of T. gondii-induced, IFN-dependent regulated necrosis to emerge (Figure 3).
