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IRG- and GBP-specific virulence mechanisms of Toxoplasma gondii

Subject Area Parasitology and Biology of Tropical Infectious Disease Pathogens
Term from 2015 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 277942395
 
Final Report Year 2023

Final Report Abstract

Virulence differences between T. gondii strains are linked to polymorphic proteins that cooperate to inactivate individual host cell Immunity-Related GTPases (IRG proteins). We identified a new T. gondii virulence effector, ROP39, as an essential element of a novel multiprotein complex that specifically inactivates Irgb10. Direct association of ROP39 with Irgb10 inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuole membrane (PVM) of the parasite. Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. T. gondii has been shown to activate the NLRP3 inflammasome but the trigger has not yet been identified. We provide evidence that vacuolar disruption is a prerequisite for NLRP3 activation. T. gondii ROP5 and ROP18 protect the PVM and thereby inhibit inflammasome activation and IL-1b release. Besides protection of the PVM, we demonstrate an additional function of ROP5 and ROP18 for NLRP3 inhibition. We demonstrate the molecular mechanism of this inhibition includes direct interaction with GBP5. Interferon lambda (IFN-λ) protects mucosal barriers during pathogen exposure. A possible contribution of IFN-λ to T. gondii control has not been investigated so far. We demonstrate with systemic interferon lambda receptor (IFNLR1) and conditional (Villin-Cre) knockout mouse models and bone marrow chimeras of oral T. gondii infection and mouse intestinal organoids a significant impact of IFN-λ signaling in intestinal epithelial cells and neutrophils to T. gondii control in the gastrointestinal tract. Besides IRG proteins, accumulation of Guanylate-Binding Proteins (GBP proteins) at the PVM is essential for parasite control. The necessity of two families of GTPases (IRG and GBP proteins) for resistance against T. gondii and their functional inhibition by the same parasite effectors infers a potential mechanistical interdependence. We identified two heterologous IRG:GBP pairs. Specific knockouts demonstrate that loading of GBP6 and Irgb6/Irgb10 to the T. gondii-derived PVM is strictly dependent on Irgb10 and GBP5 respectively. Our results reveal an interplay of IRG and GBP proteins, a novel aspect of the IRG- and GBP- mediated control of murine T. gondii infections. The largest impact on T. gondii virulence can be attributed to the pseudokinase ROP5. The rop5 locus comprises a cluster of polymorphic genes, encoding three major isoforms, ROP5A, ROP5B and ROP5C. We demonstrate that ROP5B largely determines T. gondii virulence. ROP5B but not ROP5A or ROP5C is sufficient to maintain PVM integrity and rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. ROP5 isoforms A and C seem to play only a minor role and might have co-evolved with IRG proteins to establish infections in other subspecies of mice or other important intermediate hosts than Mus musculus. Virulence of T. gondii type I strains in wild-derived mice like CIM (Mus musculus castaneus) is controlled by polymorphic IRG proteins. Especially important is binding of Irgb2-b1 to ROP5B. We show that genetically diverse T. gondii strains from South America (SA) possess a polymorphic ROP5 Irgb2-b1 binding interface that allows escape from Irgb2-b1CIM binding and vacuolar IRG protein accumulation. Our results indicate that ROP5 has co-evolved with Irgb2-b1 to adapt to local conditions.

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