17th International Congress on Toxoplasmosis

Professor

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

Host-Parasite Interaction & Signalling

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a &quot;Trojan Horse&quot; mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Our results show that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further showed that wildtype infected DCs treated with a Shp1/2 inhibitor no longer induced the F-actin stress fiber phenotype and hypermigration. Finally, we demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

Oral presentation

Mechanism of Toxoplasma gondii dissemination using immune cells as a shuttle carriers

Toxoplasma gondii has developed efficient mechanisms to disseminate within the host, reaching virtually any tissue including immunoprivileged sites like the eye and the brain. This wide distribution is achieved by using migratory immune cells as shuttle carriers. Migratory immune cells are specialised in navigation, travelling long distances and crossing complex biological barriers, to reach any cell in the host. However, using immune cells as dissemination mechanism involves a complex balance between maintaining parasite replication and migratory fitness of the infected immune cell, which is bearing an increasing large cargo. Although molecular biological data on Toxoplasma -cell hijacking are well studied, the biophysical mechanisms underneath the trojan-horse migration are unresolved. Here, using advance microscopy techniques, custom-designed microchannels, 3D matrices and parasite and immune cell mutants we investigate the biophysical principles of locomotion of immune cells with parasite cargo. Our results show that infected immune cells are able to maintain migration, path finding and navigation properties through extraordinarily tight constrictions. Under extreme cell deformation, caused by crossing narrow and rigid constrictions, host-Toxoplasma adaptations show a dynamic actin network surrounded by a cage-like host microtubule network that ensure optimal balance between host migratory properties and mechanical resilience of the parasitophorous vacuole.

Targeted in vivo screens identify GRA12 as a strain and mouse-transcendent secreted virulence factor of Toxoplasma gondii

Toxoplasma survives in a remarkably vast repertoire of host cell types and species. This ability is granted by over 200 rhoptry and dense granule proteins secreted by the parasite during or after invasion. Most effectors have been shown to be either host- and/or parasite strain-specific, such as the ROP18 kinase which is a key virulence factor only in the context of virulent parasites infecting laboratory mouse strains. However, effector proteins required to colonise more resistant mice such as the M. m. castaneus and the M. m. musculus subspecies, or virulence factors that are shared across Toxoplasma lineages are still unknown. Here, we performed CRISPR-Cas9 in vivo screens of the Toxoplasma secretome in type I, II, III and the South American VAND isolate in mouse strains that differ in their susceptibility to infection. We identified the dense granule protein 12 (GRA12) as a prominent strain-transcendent virulence factor during acute infection in resistant and susceptible mice. A CRISPR screen in IFNγ-stimulated macrophages in vitro confirmed the importance of GRA12 for Toxoplasma survival and suggests the protein protects the parasite from cell-autonomous immune responses. We established GRA12 KO and complemented strains in the RH ΔKU80 background and in a newly created VAND ΔKU80 strain and confirmed GRA12 role as a virulence factor in resistant murine subspecies in vitro and in vivo. Lack of GRA12 results in loss of vacuolar space and ruffling of the parasitophorous vacuole membrane as observed by transmission electron microscopy. Macrophages infected with GRA12-KO parasites, but not with complemented or parental strains, undergo a IFNγ-dependent cell death with features of necrosis. GRA12 homologs of the closely related species Neospora caninum and Hammondia hammondi rescue the restriction of GRA12-KO parasites, suggesting a conserved function beyond Toxoplasma. In summary, we identified the first Toxoplasma virulence factor to date that is shared across parasite and host murine strains, and which is critical for cell-autonomous survival. Further investigation on GRA12 will elucidate a Toxoplasma core mechanism to evade the host-mediated clearance.

In vivo CRISPR screen identifies a novel microneme protein essential for Toxoplasma egress and virulence

In the lytic cycle of Toxoplasma gondii, following successful invasion and replication, parasites eventually egress from the host cell. Parasite egress is not a passive process but a strictly orchestrated event driven by the interaction of factors derived from the host and parasites. The phylum Apicomplexa possesses secretory organelles called micronemes that release their contents: microneme proteins (MICs). MICs comprise various types of proteins, such as adhesins, proteases, and perforins, that are crucial for motility, invasion, and egress. In particular, perforin-like protein 1 (PLP1) is the single known MIC essential for both egress and virulence. However, it is incompletely understood whether other MICs also play pivotal roles during infection in vivo. In this study, we have generated a small-scaled gRNA library targeting approximately 300 T. gondii genes encoding proteins predicted to be localized at the apical complex (conoid and micronemes) and pellicles (IMC and plasma membrane) by hyperLOPIT. In vivo CRISPR screen analysis revealed various apical complex/pellicle-related genes that are important for parasite virulence. Among the top hits, we discovered that a novel microneme protein, whose function had been previously unknown, plays an essential role in the induction of egress by permeabilizing and disrupting both parasitophorous vacuole membrane and host plasma membrane. Moreover, we found that PLP1 alone is insufficient for parasite egress and that this novel MIC is also required. Collectively, our in vivo CRISPR screen provides a crucial clue to understanding the molecular mechanism of how MICs orchestrate parasite egress that contributes to the parasite in vivo adaptation and virulence.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons as Models for Toxoplasma gondii Infection and Persistence

Toxoplasma gondii infection may be asymptomatic in healthy people but results in the lifelong persistence of cysts containing bradyzoites in brain and muscle. We used human induced pluripotent stem cell-derived cardiomyocytes (iCMs), cardiac fibroblasts (iCFs) and neurons (iNEs) from a single human donor to study the interactions of T. gondii with human cells in vitro. iCMs, iCFs and iNEs infected with the T. gondii TypeI/III EGS reporter strain expressing SAG1-mCherry (tachyzoite) and LDH2-sfGFP (bradyzoite) displayed higher rates of bradyzoite formation in iCMs and iNEs, compared to iCFs from the same donor at 72 hours post-infection by fluorescence microscopy of live cells and flow cytometry. Bradyzoite induction occurred spontaneously in normal media. Using RNA-seq we compared uninfected iCMs, iCFs and iNEs to cells infected for 24 hours and 72 hours. Both iCFs and iCMs showed thousands of differentially expressed genes (DEGs) at both timepoints. In contrast, iNEs infected for 24 hours showed 190 upregulated and 321 downregulated DEGs whereas iNEs infected for 72 hours showed 244 upregulated and 91 downregulated DEGs. Enrichment analysis of gene sets (GSEA) regulated by infection was performed using the hallmark gene set collection from the Molecular Signatures Database. To correlate the higher rate of bradyzoite formation in iCMs and iNEs to the relevance of each GSEA hallmark pathway, we identified hallmark pathways having enriched DEGs in a similar manner between iCMs and iNEs but different from iCFs. At 24 hours post-infection, fatty acid metabolism and oxidative phosphorylation pathways were upregulated in iCFs but unchanged in iCMs or iNEs, whereas MYC targets pathway was downregulated in iCMs or iNEs but unchanged in iCFs. At 72 hours post-infection, glycolysis pathway was downregulated in iCMs or iNEs but unchanged in iCFs, whereas myogenesis and protein secretion pathways were upregulated in iCFs but were unchanged in iCMs or iNEs. Our in vitro generated human iCMs, iCFs and iNEs provide a new model to understand the impact of host cell type and host-specific factors upon Toxoplasma infection. We anticipate this model can be used to develop cell-based assays for drug screening of potential bradyzoite therapeutic targets and to explore the role of host genetics in T. gondii pathogenesis and persistence.

Toxoplasma gondii restricts the developmental trajectory of placental trophoblasts from an infection-resistant cell fate

Pregnancy is one of the most critical points of vulnerability to Toxoplasma infection. Despite this, no specific mechanisms used by the parasite to cross the placenta have been discovered. Here, we investigate an entirely new phenomenon where Toxoplasma is able to coopt canonical host cell development to propagate its own transmission through the placenta. Using a bipotent stem cell system, we can grow cells that recapitulate placental trophoblast progenitors, called cytotrophoblasts (CYT), in addition to both terminally differentiated lineages, extravillous trophoblasts (EVT) and syncytiotrophoblasts (SYN). These cells vary in their susceptibility to T. gondii infection, with SYN being unusually resistant and EVT being comparably permissive. Importantly, the high proportion of infection-resistant SYN at the surface of the maternofetal interface prevents the vast majority of parasite invasion to deeper cell layers. Using scRNA and scATAC-seq datasets of 50,000 cells, we have convincingly determined that T. gondii-infected progenitor CYT undergo global transcriptional changes consistent with a departure from their stem state and activation of host cell differentiation. Moreover, these data indicate that this parasite-induced host cell differentiation is specifically antagonistic to the infection-resistant SYN cell lineage. We see simultaneous reduction of transcripts and proteins governing SYN development and induction of transcripts governing EVT development. We also demonstrate that parasite infection of 3D primary trophoblast organoids (a system that best recapitulates inter-cell type signaling) for just 24 hours is sufficient to elicit CYT differentiation to EVT-like cells that, ordinarily, must be chemically induced for multiple days in culture. Finally, we use a novel functional assay to show that Toxoplasma infection results in such severe lineage restriction that host cells almost completely lose their capacity to fuse with other SYN. Taken together, these results indicate that Toxoplasma infection dysregulates progenitor trophoblast cells at both the levels of cell fate and cell function, meaning, infection of progenitor cells specifically promotes one cell lineage at the cost of another. This significant shift of CYT away from SYN and towards EVT could alter the balance of resistant and susceptible cells at the maternofetal interface, potentiating robust T. gondii dissemination through placental tissue and, ultimately, into the fetal compartment.

Session X: Parasite-Host Interactions & Signalling II

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