Molecular Interactions of Toxoplasma gondii Dense Granule 23 (GRA23) with Host Proteins PEX3 and TRAP1

Xuewei Fan¹Nan Zhang²Xichen Zhang²Shudong Li¹Jiangming Fu¹Min Sun²Xiaoxiao Ma²Muhammad Zahoor Khan³Xin Jiang^1*

• ¹Heilongjiang Vocational College of Agricultural Economics, Mudanjiang, China
• ²College of Veterinary Medicine, Jilin University, Changchun, Jilin, China
• ³Liaocheng University, Liaocheng, Shandong Province, China

Toxoplasma gondii, an obligate intracellular protozoan, utilizes dense gran-ule proteins to modulate host cell processes. Dense granule protein 23 (GRA23) facilitates molecular trafficking between the parasitophorous vacuole and host cell cyto-plasm, though its specific host protein interactions remain poorly characterized. This study employed pull-down assays coupled with mass spectrometry to identify host proteins interacting with GRA23. Among 35 proteins identified, peroxisomal biogenesis factor 3 (PEX3) and TNF receptor-associated protein 1 (TRAP1) were validated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significant enrichment of these interacting proteins in metabolic pathways and cellular processes related to reproduction, growth, and development. The interaction between GRA23 and PEX3 suggests potential parasite modulation of peroxisomal functions, while its association with TRAP1 indicates possible exploitation of host chaperone mechanisms. This study provides the first evidence that GRA23 interacts with host proteins implicated in key cellular functions, offering novel insights into T. gondii pathogenesis and potential therapeutic targets for toxoplasmosis treatment.