The Regulation of Plasmodium falciparum Metabolism by Haloacid Dehalogenase Proteins

Malaria is an enormous financial and public health burden for much of the world, infecting over 200 million and killing over 400,000 people every year. While much progress has been made combating malaria in the past few decades, those advances have slowed in recent years, partially due to the emergence of resistance to all known antimalarials used to date. To achieve the goal of eliminating malaria as a major global health problem, new therapeutics need to be developed, targeting novel categories of parasite biology. One poorly understood area of parasite biology is the regulation of various metabolic pathways. We have recently identified a superfamily of proteins, named haloacid dehalogenase (HAD) proteins, that are implicated in resistance to metabolic inhibitors and regulation of essential metabolic pathways in Plasmodium falciparum malaria parasites. Here, we investigate how HAD2 (PF3D7_1226300) regulates metabolism of the isoprenoid biosynthesis pathway, using biochemical, metabolomic, and genetic tools. We find that HAD2 is a phosphatase with a preference for triose phosphates. We then investigate the related HAD proteins—HAD4 (PF3D7_1118400), Lipin (PF3D7_0303200), and HAD5 (PF53D7_1017400)—for their roles in regulating parasite metabolism and the implications for future drug design. We find that HAD4 and Lipin are dispensable for growth in asexual malaria parasites. Lipin disruption causes significant growth reduction and accumulation of lipid species, while HAD4 is a dispensable nucleotide phosphatase. We also find that HAD5 is a phosphomannomutase that is essential for parasite egress and invasion. We solve the three-dimensional crystal structure of HAD5 and demonstrate our ability to selectively inhibit it compared to human phosphomannomutases. All of these findings add to our understanding of metabolic regulation in malaria parasites, illuminating key ways that targeting different metabolic pathways could work synergistically in development of novel antimalarial therapeutic strategies

Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiple

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target

Haloacid Dehalogenase Proteins: Novel Mediators of Metabolic Plasticity in

Widespread antimalarial drug resistance has prompted the need for new therapeutics and greater understanding of malaria parasite biology. To this end, the isoprenoid biosynthesis inhibitor fosmidomycin has been used to probe the metabolic regulation in the malaria parasite, Plasmodium falciparum . Genetic changes in the haloacid dehalogenase (HAD) superfamily member HAD2 conferred resistance to fosmidomycin, at the cost of decreased fitness. In the absence of fosmidomycin, parasites gained mutations to phosphofructokinase that restored growth and fosmidomycin sensitivity, thus revealing an intriguing example of plasticity in a core glycolytic process. Moreover, this study marks a second report of a HAD superfamily protein-modulating metabolic homeostasis in P falciparum parasites. Haloacid dehalogenase enzymes are distributed across all domains of life and have increasingly been found to influence central carbon metabolism and drug sensitivity in P falciparum . Investigating the mechanisms by which HAD superfamily members modulate metabolism may shed light on how metabolic networks are connected in apicomplexan parasites and other organisms and may guide future therapeutic endeavors

Suppression of Drug Resistance Reveals a Genetic Mechanism of Metabolic Plasticity in Malaria Parasites

ABSTRACT In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identified a loss-of-function mutation in HAD2 (P. falciparum 3D7_1226300 [PF3D7_1226300]) as necessary for FSM resistance. Enzymatic characterization revealed that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we selected for suppression of HAD2-mediated FSM resistance and uncovered hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase 9 (PFK9). Metabolic profiling demonstrated that FSM resistance is achieved via increased steady-state levels of methylerythritol phosphate (MEP) pathway and glycolytic intermediates and confirmed reduced PFK9 function in the suppressed strains. We identified HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncovered PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our report informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation. IMPORTANCE Unique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms is urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our report identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncovered mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance of antimalarials