Toxoplasma GRA72 is Involved in Membrane-Associated Functions and Metabolic Homeostasis Revealed by Transcriptomic Analysis.

BACKGROUND: Toxoplasma gondii is a widely disseminated obligate intracellular parasite, infecting approximately one-third of the world human population. While infections in healthy individuals are typically asymptomatic or mild, the parasite poses a serious threat to specific populations. Immunocompromised individuals (such as AIDS patients and organ transplant recipients) risk life-threatening toxoplasmic encephalitis, and congenital infection during pregnancy can lead to fetal loss or severe neurological and ocular defects in newborns. GRA72, one of the dense granule proteins secreted by T. gondii, is known to localize to the parasitophorous vacuole membrane (PVM) and parasitophorous vacuole (PV). Though important for PVM permeability, PV morphology, tachyzoite growth, and virulence, GRA72’s imapct on the global parasite transcription and its potential interacting or compensatory proteins remains unclear. METHOD: High-throughput RNA sequencing (RNA-seq) was performed on the pre-constructed T. gondii GRA72 gene knockout strain (PruΔgra72) and wild-type Pru strain to compare differences in global gene expression. Differentially expressed genes (DEGs) were identified and subjected to functional annotation and pathway enrichment analysis. Gene Ontology (GO) enrichment analysis were conducted based on GO annotation and the standard classification system. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was utilized to identify major metabolic and signaling pathways associated with these DEGs. RNA-seq results were verified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: RNA-seq analysis identified 858 DEGs in PruΔgra72 strain compared to the wild-type strain, comprising 550 upregulated and 308 downregulated. Upregulation of bradyzoite associated genes were observed in PruΔgra72, while expression of dense granule proteins (GRAs) exhibited bidirectional regulation. RT-qPCR analysis validated the RNA-seq data. GO enrichment analysis revealed significantly enrichment of DEGs in membrane-related functional categories. KEGG pathway analysis indicated that the DEGs were significantly enriched in key metabolic pathways, including fatty acid, carbon and amino acid metabolism. CONCLUSION: This study provide a new perspective for studying the pathogenic mechanisms of T. gondii, revealing that GRA72 modulates membrane-associated functions and metabolic homeostasis. The observed transcriptional changes, particularly the upregulation of bradyzoite genes and altered expression of GRAs, suggest that GRA72 may play an important role in parasite development. Further investigation of the compensatory or interacting upstream/downstream genes associated with membrane function and reprogrammed metabolism (fatty acid, carbon, amino acid) will elucidate GRA72’s molecular mechanisms and could facilitate the development of novel anti-Toxoplasma therapeutics.