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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Toxoplasma sensing (Cytoplasmic sensing of Toxoplasma gondii infection and host cell programmed necrosis)

Teaser

Summary

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 â€“ 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 (IFNÎ³) limits parasite dissemination in the host and drives (asymptomatic) latency.
My focus was to gain a greater understanding of events taking place in IFNÎ³-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

Work performed

With respect to Aim 1, I found a role for galectin-8 (and galectin-9) in Toxoplasma sensing, a work that is close to being submitted for publication. Furthermore, ongoing work screening PRRs - which may still yield additional sensor(s) - has led to exclusion of many candidate genes, and will also be published. As for aim 2, short of identifying the pathway by which fibroblasts die in response to avirulent T. gondii, I have diligently eliminated several candidate genes and mechanisms. I found that the necrosis response to T. gondii does not require the RIPK3-MLKL-RIPK1-CASP8 necrosome and therefore does not proceed through the canonical necroptosis patway. This work and preliminary evidence suggesting a role for ASC and inflammasomes is nearing completion/publication. I envision discovery of the â€œculpritâ€ genes and additional publications is imminent.
I developed 2 novel assays â€“ one based on flow cytometry or on live-cell fluorescence microscopy â€“ that provide multi-parametric information, crucial insights, and have been the basis for faster progress for myself and others in the lab. A manuscript containing the description of the 2 methods was published and will be of benefit to other researchers.
I attended and presented posters at a Keystone Symposium on Nucleic Acid Sensing Pathways in 2016 and at the International Toxoplasma Congress in 2017 and 2019, attended several courses and workshops, and discussed my MSCA-funded research in numerous outreach activities, namely with schools and lay audiences, as well as via the Improvisational Theatre group that I founded called 84Po in honor of Marie Curie\'s discovery of Polonium.

Final results

Virtually nothing was known about Toxoplasma sensing or the molecular mechanisms underlying host cell necrosis. My work showed that most of the cytosolic PRRs that detect pathogen DNA/RNA play but moderate roles in Toxoplasma sensing. I found that galectins, in particular galectin-8 (and -9), function as PVM damage receptors in murine cells. Through painstaking elimination of possible candidates, I found that the canonical pathway of regulated necrosis, necroptosis, is dispensable. That many of the usual suspects could not be implicated may have made the road to finding the sensing/necrosis â€œculpritsâ€ arduous but, encouragingly, solidifies the notion that this a non-canonical process. Toxoplasma infection, therefore, provides us with a privileged insight into yet uncharacterized regulated necrosis pathways that, despite being paramount to limiting parasite, bacteria and viral infection and driving inflammatory responses, are still in dire need of mechanistic clarification. Pharmacological manipulation of necroptosis, inflammasomes, lysosomal permeability or cathepsins yields great promise in sensitization of cancer cells to chemo and radiotherapy or in inflammatory disease treatment. However, harnessing the therapeutic intervention potential of pathways involved in pathogen sensing and necrosis requires more accurate knowledge of the underlying mechanisms. I believe this MSCA has provided crucial insights into both of these processes.