Unraveling the role of glucosamine: Fructose-6-phosphate aminotransferase (GFAT) in Entamoeba histolytica: A comprehensive exploration through biochemical, reverse genetic, and Metabolomic analyses.

Entamoeba histolytica is a protozoan parasite that causes amebiasis in humans. Its life cycle consisting of two stages: the proliferative trophozoite, which is responsible for clinical symptoms, and the dormant cyst, which mediates disease transmission. The cyst wall is primarily composed of chitin, a polymer of β-1,4-linked N-acetylglucosamine. The hexosamine biosynthetic pathway (HBP) is crucial for producing uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), an essential metabolite for chitin and glycoprotein synthesis. Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the rate-limiting step of this pathway; however, its role in E. histolytica, particularly during trophozoite growth and cyst conversion, remains unclear. In this study, we combined biochemical, reverse genetic, and metabolomic approaches to investigate GFAT function. Epigenetic silencing of the EhGFAT gene reduced intracellular glucosamine-6-phosphate (GlcN6P) levels, decreased N-linked glycosylation of EhCP-5 A, and moderately inhibited trophozoite growth. Treatment with the GFAT inhibitor 6-diazo-5-oxo-L-norleucine (DON) inhibited encystation in the model species E. invadens in a dose-dependent manner. Global metabolomic profiling revealed that EhGFAT silencing reduced metabolites involved in hexosamine metabolism (e.g., GlcN6P and UDP-GlcNAc) and altered amino acid and energy metabolism, including ornithine, proline, arginine, fumarate, and succinate. In contrast, metabolites from the de novo cysteine biosynthetic pathway, such as O-acetylserine and S-methylcysteine were increased. Our findings demonstrate that GFAT plays an important role in the hexosamine biosynthetic pathway, trophozoite proliferation, and efficient encystation in Entamoeba. While the precise molecular mechanisms underlying these processes require further investigation, this study provides new insights into GFAT function and supports its further evaluation as a potential therapeutic target for amebiasis.