Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins secreted from dense granule organelles to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions, we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant "bubble vacuole" morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCE: Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant 'bubble vacuole' morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections.

Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.

Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.

CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells.

Toxoplasma virulence depends on its ability to evade or survive the toxoplasmacidal mechanisms induced by interferon gamma (IFNγ). While many Toxoplasma genes involved in the evasion of the murine IFNγ response have been identified, genes required to survive the human IFNγ response are largely unknown. In this study, we used a genome-wide loss-of-function screen to identify Toxoplasma genes important for parasite fitness in IFNγ-stimulated primary human fibroblasts. We generated gene knockouts for the top six hits from the screen and confirmed their importance for parasite growth in IFNγ-stimulated human fibroblasts. Of these six genes, three have homology to GRA32, localize to dense granules, and coimmunoprecipitate with each other and GRA32, suggesting they might form a complex. Deletion of individual members of this complex leads to early parasite egress in IFNγ-stimulated cells. Thus, prevention of early egress is an important Toxoplasma fitness determinant in IFNγ-stimulated human cells. IMPORTANCE Toxoplasma infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth. While most Toxoplasma parasites are cleared by this immune response, some can survive by blocking or evading the IFNγ-induced restrictive environment. Many Toxoplasma genes that determine parasite survival in IFNγ-activated murine cells are known but parasite genes conferring fitness in IFNγ-activated human cells are largely unknown. Using a Toxoplasma adapted genome-wide loss-of-function screen, we identified many Toxoplasma genes that determine parasite fitness in IFNγ-activated human cells. The gene products of four top hits play a role in preventing early parasite egress in IFNγ-stimulated human cells. Understanding how IFNγ-stimulated human cells inhibit Toxoplasma growth and how Toxoplasma counteracts this, could lead to the development of novel therapeutics.

TDR Targets 6: driving drug discovery for human pathogens through intensive chemogenomic data integration.

The volume of biological, chemical and functional data deposited in the public domain is growing rapidly, thanks to next generation sequencing and highly-automated screening technologies. These datasets represent invaluable resources for drug discovery, particularly for less studied neglected disease pathogens. To leverage these datasets, smart and intensive data integration is required to guide computational inferences across diverse organisms. The TDR Targets chemogenomics resource integrates genomic data from human pathogens and model organisms along with information on bioactive compounds and their annotated activities. This report highlights the latest updates on the available data and functionality in TDR Targets 6. Based on chemogenomic network models providing links between inhibitors and targets, the database now incorporates network-driven target prioritizations, and novel visualizations of network subgraphs displaying chemical- and target-similarity neighborhoods along with associated target-compound bioactivity links. Available data can be browsed and queried through a new user interface, that allow users to perform prioritizations of protein targets and chemical inhibitors. As such, TDR Targets now facilitates the investigation of drug repurposing against pathogen targets, which can potentially help in identifying candidate targets for bioactive compounds with previously unknown targets. TDR Targets is available at https://tdrtargets.org.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells.

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals. This parasite is an obligate intracellular pathogen and actively invades susceptible host cells, a process which is mediated by specific receptor-ligand interactions. Here, we have identified an unnatural 2,4-disulfated d-glucuronic acid (Di-S-GlcA), a hexuronic acid composed of heparin/heparan sulfate, as a potential carbohydrate ligand that can selectively bind to T. gondii parasites. More importantly, the gelatin conjugated Di-S-GlcA multivalent probe displayed strong inhibition of parasite entry into host cells. These results open perspective for the future use of Di-S-GlcA epitopes in biomedical applications against toxoplasmosis.