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.

Keywords: Plasmodium; antimalarial agents; drug resistance mechanisms; fosmidomycin; glycolysis; isoprenoids; malaria; metabolic regulation; metabolism.

FIG 1

FSM^r strain E2 possesses a mutation in HAD2, a homolog of MEP pathway regulator HAD1. (A) Representative FSM dose response of the parental strain and strain E2. (B) P. vivax HAD2 (teal; PDB 2B30) is structurally similar to PfHAD1 (gray; PDB 4QJB). Ions (Mg²⁺, Ca²⁺, Cl^-) are shown in yellow. (C) HAD2 is a homolog of HAD1 (29% identity and 53% similarity) and possesses all conserved HAD sequence motifs required for catalysis (37).

FIG 2

PfHAD2 is a phosphatase. (A) HAD2 is an active phosphatase, and HAD2^D26A is a catalytic mutant (cat. mut.) that can be used as a negative control for HAD2-specific activity. “No enz.” represents a no-enzyme control. Data shown represent the enzyme activities seen using the synthetic phosphatase substrate pNPP. Error bars represent standard errors of the means (SEM) (****, P ≤ 0.0001 [unpaired t test]; n.s., not significant). (B) Activity of HAD2, normalized to the activity of the catalytic mutant (HAD2^D26A), for a variety of substrates (2-GlcP, 2-glycerol-phosphate; M6P, mannose-6-phosphate; FBP, fructose-2,6-bisphosphate; dAMP, deoxy-AMP). Error bars represent SEM.

FIG 3

Leveraging resistance-associated growth attenuation to identify genetic changes that modulate FSM sensitivity. (A) Prolonged culture resulted in loss of FSM resistance in strain E2. Shown are FSM dose responses of the strain E2 before (day 9) and after (day 79) prolonged culture without FSM. Nine days after thawing resistant strain E2, we observed an FSM IC₅₀ of 4.9 μM, while after 79 days of culture without FSM, E2 had an FSM IC₅₀ of 1.3 μM. The dose responses were part of routine evaluation of individual strain phenotypes at discrete points in time. Each data point is representative of the mean from two technical replicates. Error bars represent SEM. (B) Parasites are colored according to FSM phenotype (teal, FSM^s; purple, FSM^r). Cloned strains are named according to FSM phenotype (E2-SX, sensitive; E2-RX, resistant). An FSM^s parental strain was selected under conditions of FSM pressure to enrich for FSM^r strain E2 (had2^R157X). After relief of FSM pressure, a fitness advantage selected for spontaneous suppressor mutations in PFK9 (pfk9^mut; yellow star) that resulted in FSM sensitivity. FSM^r clones were grown without FSM pressure, and a fitness advantage again selected for suppressor mutations in PFK9 that resulted in an increased growth rate and loss of FSM resistance.

FIG 4

Suppressor strains with PFK9 mutations display changes in FSM tolerance and growth. (A) Suppressed clones have significantly lower FSM IC₅₀s (****, P ≤ 0.0001). Error bars represent SEM. HAD2 and PFK9 genotypes for each strain are indicated. For reference, the parental (par) strain data are shown in the black column. All data are representative of results from ≥3 independent experiments. (B) FSM resistance results in a fitness cost. FSM^r clones with the had2^R157X allele (R1 to R3, purple lines) had reduced growth rates compared to the wild-type parental (par) strain (black) (*, P ≤ 0.05). The growth defect was rescued in clones with mutations in PFK9 (S1 to S5, teal lines). Growth was normalized to parasitemia on day 0. Error bars represent SEM of results from ≥3 independent growth experiments.

FIG 5

Loss of HAD2 is necessary for FSM resistance. (A) Successful expression of pTEOE110:HAD2-GFP in strain R2 (had2^R157X, PFK9) was confirmed by immunoblotting. Marker units are indicated in kilodaltons (kDa). The top blot was probed with anti-HAD2 antiserum (expected masses: HAD, 33 kDa; HAD2-GFP, 60 kDa). The bottom blot was probed with anti-heat shock protein 70 (Hsp70) antiserum as a loading control. (B) Representative FSM dose response demonstrating that expression of HAD2-GFP in strain R2 (had2^R157X PFK9) resulted in restored sensitivity to FSM. Strain R2 had an elevated FSM IC₅₀ compared to the parental (par) strain. When HAD2 expression was restored in strain R2, the resulting strain showed an IC₅₀ near that of the parent strain. Data shown are from a representative clone (clone 1) of the HAD2-rescued strain. Additional clones displayed a similar phenotype (see Fig. S2).

FIG 6

PFK9 alleles in suppressed strains are hypomorphic. (A) Schematic of suppressor mutations found in PFK9. Strain names and resulting amino acid changes are indicated. Three of the four mutations are found on the structural model of PfPFK. The parts of the protein represented by the model are notated by the teal arrows under the α and β domains. The total protein length is 1,418 amino acids. N1359Y fits outside the model. The other mutations are represented by their stick model structure, with the resulting change shown in magenta. Orientations of the closeup representations of the mutations are indicated where they differ from the main model. (B) Measurement of PFK activity of P. falciparum lysate indicated that E2-SX clones with PFK9 suppressor mutations have significantly reduced PFK activity (****, P ≤ 0.0001 [ANOVA, Sidak’s posttest]). Error bars represent SEM. Assay data are linear with respect to protein content and specific for PfPFK9 activity (Fig. S3).

FIG 7

HAD2 and PFK9 alleles alter FSM resistance and metabolite levels in P. falciparum. (A) Metabolic profiling and clustering of parental (par) and E2 clone strains demonstrated a metabolic signature of resistance, which included increased levels of MEP pathway intermediates DOXP and MEcPP and the glycolytic metabolite and PFK product FBP. Glu6P/fru6P and DHAP/gly3P are isomer pairs that cannot be confidently distinguished. Clustering was performed using the heatmap function in R. Data are also summarized in Table S1 and Fig. S5. FSM IC₅₀s are shown for reference. FC, fold change. (B) DOXP levels were highly correlated to levels of the upstream glycolytic metabolic FBP (Pearson’s r = 0.95). (C) By contrast, DOXP levels were not correlated to the glycolytic metabolites glu6P/fru6P (Pearson’s r = 0.57).

FIG 8

Model of HAD2- and PFK9-mediated metabolic regulation. Abbreviations: fru6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; gly3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; pyr, pyruvate; PEP, phosphoenolpyruvate; DOXP, deoxyxylulose 5-phosphate, MEcPP, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate; IPP, isopentenyl pyrophosphate. Black circles represent metabolites. Key metabolites (FBP, DOXP, and MEcPP) are shown in purple. The glycolytic metabolites DHAP and PEP are imported into the apicoplast and are converted to gly3P and pyruvate, respectively (89, 90). HAD2 may act as a negative regulator of PFK9, directly or indirectly, or may inhibit glycolysis downstream of PFK9. Loss of HAD2 results in increased substrate availability to the MEP pathway. In had2 strains, reduction in PFK9 activity may counteract or bypass metabolic perturbations due to loss of HAD2.