Fenollar, A. | bioRxiv Science

bioRxiv2024

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.

microbiology↗

bioRxiv2023

Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

A promising treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation, by competition. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional gene knockouts were created to investigate the biological importance of the FOH/GGOH salvage pathway. The knockout parasites were viable but more susceptible to fosmidomycin, and their sensitivity to MEP inhibitors could not be rescued by the addition of prenols. Moreover, the knockout parasites lost their ability to use prenols for protein prenylation. These results demonstrate that FOH/GGOH salvage is an additional source of isoprenoids by malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme whose inhibition may potentiate the antimalarial efficacy of drugs that affect isoprenoid metabolism.

biochemistry↗