Abstract
Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparumIMPORTANCE Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway.
Highlights
Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway
We demonstrate that primary sulfonamide (PS)-3 resistance in P. falciparum is not linked to the PS moiety, as no change in the resistance profile was observed for PS-3=, the non-PS structural analogue of PS-3 (Table 2)
These data support genome sequencing data of 3D7-C3PS3 clones and subsequent validation using reverse genetics, which indicate that the point mutation (R860G) in PfPFK9, the gene that encodes an isoform of the glycolytic enzyme phosphofructokinase, contributes to PS-3 resistance
Summary
Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. We show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Fosmidomycin, an antibiotic and antimalarial drug candidate, acts as a competitive inhibitor of a key enzyme in methylerythritol phosphate (MEP) pathway isoprenoid biosynthesis in the apicoplast of the malaria parasite [8]. In fosmidomycin-resistant parasites, loss of PfHAD1 function results in dysregulation of glycolysis and increased flux of triose-phosphates into the MEP pathway, with a concomitant reduction in the effectiveness of fosmidomycin [9]. These PfHAD2 mutants gain mutations in PfPFK9 that result in the restoration of fosmidomycin sensitivity [7]
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