Abstract
In the malaria parasite Plasmodium falciparum, isoprenoid synthesis from glycolytic intermediates is essential for survival. The antibiotic and antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In FSM-resistant P. falciparum, we identify a loss-of-function mutation in HAD2 as causative for resistance. Enzymatic characterization shows that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, functions as a nucleotidase. Harnessing a growth defect in HAD2-mutant parasites, we select for suppression of HAD2-mediated FSM resistance and uncover hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase. Metabolic profiling demonstrates that FSM resistance is achieved via increased steady-state levels of MEP pathway and glycolytic intermediates and confirms reduced PFK9 function in the suppressed strains. We identify HAD2 as a novel regulator of malaria glycolytic metabolism and drug sensitivity. Our study informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveal a previously undescribed strategy for cellular glycolytic regulation.