Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Pyrimethamine 3D printlets for pediatric toxoplasmosis: design, pharmacokinetics, and anti-toxoplasma activity.

OBJECTIVES: The focus of the present research is to develop printlet formulations of pyrimethamine (PMT). METHODS: Printlets formulation of PMT were developed by screening design by varying laser scanning speed, Kollidon® VA 64, polyvinylpyrrolidone, and disintegrant. RESULTS: Laser scanning speed, Kollidon® VA, and disintegrant had statistically significant effect on hardness, disintegration time, and/or dissolution (p < 0.05). Dissolution was almost 100% in 30 min. X-ray powder diffraction indicated partial amorphous transformation of the crystalline drug. Pharmacokinetic and anti-toxoplasma activity profiles of the printlets and compressed tablets were superimposable with no statistical difference (p > 0.05). CONCLUSION: Clinical performance of the printlets would be similar to the compressed tablets.

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages.

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.

In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration.

Toxoplasma can reach distant organs, especially the brain, leading to a lifelong chronic phase. However, genes involved in related in vivo processes are currently unknown. Here, we use focused CRISPR libraries to identify Toxoplasma genes that affect in vivo fitness. We focus on TgWIP, whose deletion affects Toxoplasma dissemination to distant organs. We show that TgWIP is secreted into the host cell upon invasion and interacts with the host WAVE regulatory complex and SHP2 phosphatase, both of which regulate actin dynamics. TgWIP affects the morphology of dendritic cells and mediates the dissolution of podosomes, which dendritic cells use to adhere to extracellular matrix. TgWIP enhances the motility and transmigration of parasitized dendritic cells, likely explaining its effect on in vivo fitness. Our results provide a framework for systemic identification of Toxoplasma genes with in vivo effects at the site of infection or on dissemination to distant organs, including the brain.

Toxoplasma GRA15 Activates the NF-κB Pathway through Interactions with TNF Receptor-Associated Factors.

The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.

Three Toxoplasma gondii Dense Granule Proteins Are Required for Induction of Lewis Rat Macrophage Pyroptosis.

Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.IMPORTANCE Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite's replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite.