Hexosamine Biosynthesis Disruption Impairs GPI Production and Arrests Plasmodium falciparum Growth at Schizont Stages

UDP-N-acetylglucosamine (UDP-GlcNAc) is a crucial sugar nucleotide for glycan synthesis in eukaryotes. In the malaria parasite Plasmodium falciparum, UDP-GlcNAc is synthesized via the hexosamine biosynthetic pathway (HBP) and is essential for glycosylphosphatidylinositol (GPI) anchor production, the most prominent form of protein glycosylation in the parasite. In this study, we explore a conditional knockout of glucosamine-6-phosphate N-acetyltransferase (PfGNA1), a key HBP enzyme. PfGNA1 depletion led to significant disruptions in HBP metabolites, impairing GPI biosynthesis and causing mislocalization of the merozoite surface protein 1 (MSP1), the most abundant GPI-anchored protein in the parasite. Furthermore, parasites were arrested at the schizont stage, exhibiting severe segmentation defects and an incomplete rupture of the parasitophorous vacuole membrane (PVM), preventing egress from host red blood cells. Our findings demonstrate the critical role of HBP and GPI biosynthesis in P. falciparum asexual blood stage development and underscore the potential of targeting these pathways as a therapeutic strategy against malaria. Author SummaryMalaria remains a major cause of illness and death, particularly in sub-Saharan Africa, with increasing resistance to treatments highlighting the urgent need for new strategies. Malaria parasites rely on the hexosamine biosynthetic pathway to produce UDP-N-acetylglucosamine, an essential metabolite for glycosylphosphatidylinositol synthesis. Glycosylphosphatidylinositol molecules anchor vital proteins to the parasites surface and, as free glycolipids, serve as structural components of its membranes. Our study examined the effects of disrupting PfGNA1, a key enzyme in the hexosamine biosynthetic pathway, which is distinct from its human counterparts. Disruption of PfGNA1 blocked the production of glycosylphosphatidylinositol, leading to improper protein localization, developmental arrest, and failure of the parasites to mature or exit infected red blood cells. Our results underscore the central role of the hexosamine biosynthetic pathway and glycosylphosphatidylinositol biosynthesis, which are essential for parasite survival. This pathway represents a promising target for developing novel antimalarial therapies.