المؤلفون: Mebratu A. Bitew, Pablo S. Gaete, Christopher Swale, Parag Maru, Jorge E. Contreras, Jeroen P. J. Saeij

المصدر: mBio, Vol 15, Iss 3 (2024)

مصطلحات موضوعية: Toxoplasma gondii, GRA47, GRA72, pore, pore-lining histidine, Xenopus oocytes, Microbiology, QR1-502

الوصف: ABSTRACTToxoplasma 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.IMPORTANCEToxoplasma 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.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2150-7511Test

الوصول الحر: https://doaj.org/article/a9e7c683dca44926a28c7b2713c37571Test

المؤلفون: Shruthi Krishnamurthy, Parag Maru, Yifan Wang, Mebratu A. Bitew, Debanjan Mukhopadhyay, Yoshiki Yamaryo-Botté, Tatiana C. Paredes-Santos, Lamba O. Sangaré, Christopher Swale, Cyrille Y. Botté, Jeroen P. J. Saeij

المصدر: mBio, Vol 14, Iss 2 (2023)

مصطلحات موضوعية: CRISPR screen, Toxoplasma gondii, effector functions, host-pathogen interactions, interferons, Microbiology, QR1-502

الوصف: ABSTRACT 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.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2150-7511Test

الوصول الحر: https://doaj.org/article/aa0b609ab8e24e4ba3325ba254cf6795Test