A High-Throughput Amenable Dual Luciferase System for Measuring Toxoplasma gondii Bradyzoite Viability after Drug Treatment.

It is estimated that more than 2 billion people are chronically infected with the intracellular protozoan parasite Toxoplasma gondii (T. gondii). Despite this, there is currently no vaccine to prevent infection in humans, and there is no recognized curative treatment to clear tissue cysts. A major hurdle for identifying effective drug candidates against chronic-stage cysts has been the low throughput of existing in vitro assays for testing the survival of bradyzoites. We have developed a luciferase-based platform for specifically determining bradyzoite survival within in vitro cysts in a 96-well plate format. We engineered a cystogenic type II T. gondii PruΔku80Δhxgpr strain for stage-specific expression of firefly luciferase in the cytosol of bradyzoites and nanoluciferase for secretion into the lumen of the cyst (DuaLuc strain). Using this DuaLuc strain, we found that the ratio of firefly luciferase to nanoluciferase decreased upon treatment with atovaquone or LHVS, two compounds that are known to compromise bradyzoite viability. The 96-well format allowed us to test several additional compounds and generate dose-response curves for calculation of EC50 values indicating relative effectiveness of a compound. Accordingly, this DuaLuc system should be suitable for screening libraries of diverse compounds and defining the potency of hits or other compounds with a putative antibradyzoite activity.

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins.

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts.

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination.

Toxoplasma gondii is an obligate intracellular protozoan with the ability to infect virtually any type of nucleated cell in warm-blooded vertebrates including humans. Toxoplasma gondii invades immune cells, which the parasite employs as shuttles for dissemination by a Trojan horse mechanism. Recent findings are starting to unveil how this parasite orchestrates the subversion of the migratory functions of parasitised mononuclear phagocytes, especially dendritic cells (DCs) and monocytes. Here, we focus on how T. gondii impacts host cell signalling that regulates leukocyte motility and systemic migration in tissues. Shortly after active parasite invasion, DCs undergo mesenchymal-to-amoeboid transition and adopt a high-speed amoeboid mode of motility. To trigger migratory activation - termed hypermigratory phenotype - T. gondii induces GABAergic signalling, which results in calcium fluxes mediated by voltage-gated calcium channels in parasitised DCs and brain microglia. Additionally, a TIMP-1-CD63-ITGB1-FAK signalling axis and signalling via the receptor tyrosine kinase MET promotes sustained hypermigration of parasitised DCs. Recent reports show that the activated signalling pathways converge on the small GTPase Ras to activate the MAPK Erk signalling cascade, a central regulator of cell motility. To date, three T. gondii-derived putative effector molecules have been linked to hypermigration: Tg14-3-3, TgWIP and ROP17. Here, we discuss their impact on the hypermigratory phenotype of phagocytes. Altogether, the emerging concept suggests that T. gondii induces metastasis-like migratory properties in parasitised mononuclear phagocytes to promote infection-related dissemination.

Convergent Met and voltage-gated Ca2+ channel signaling drives hypermigration of Toxoplasma-infected dendritic cells.

Ras-Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. However, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We report that signaling through the receptor tyrosine kinase Met (also known as HGF receptor) contributes to T. gondii-induced DC hypermotility. Furthermore, voltage-gated Ca2+ channel (VGCC, subtype CaV1.3) signaling impacted the migratory activation of DCs via calmodulin-calmodulin kinase II. We show that convergent VGCC signaling and Met signaling activate the GTPase Ras to drive Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively) phosphorylation and hypermotility of T. gondii-infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. This emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties that promote infection-related dissemination.

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.

Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii Further, gene silencing and antibody blockade of the ß1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1-CD63-ITGB1-FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.

Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix.

Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a "Trojan horse" mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T. gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naïve DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination.

Voltage-dependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection, γ-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.