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

Abstract

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.

Keywords: carbohydrate metabolism; crystal structure; drug development; glycosylphosphatidylinositol (GPI) anchor; haloacid dehalogenase (HAD); malaria; parasite; phosphomannomutase.

Figure 1

HAD5 is a bifunctional phosphomannomutase/phosphoglucomutase.A, schematic of phosphomannomutases’ role in metabolism. Phosphomannomutases like HAD5 interconvert mannose 6-phosphate and mannose 1-phosphate, providing the latter for downstream glycolipid production and synthesis of GPI anchors in P. falciparum. B, SDS-PAGE gel of recombinant WT HAD5 and a catalytically inactive mutant (D11A). C, displayed are the mean ± SEM of HAD5 activity across three independent trials, with D11A activity subtracted as background. p-values were determined using an ordinary two-way ANOVA (Tukey's test for multiple comparisons, α = 0.05). ∗∗∗∗p < 0.0001, ns = not significant. G-1,6-P, glucose-1,6-bisphosphate; GPI, glycosylphosphatidylinositol; PGM, phosphoglucomutase assay; PMM, phosphomannomutase assay.

Figure 2

HAD5 is essential for intraerythrocytic parasite growth.A, schematic of the regulatable knockdown system introduced at the native locus of PfHAD5 (46, 47). B, Western blot of transgenic HAD5^KD parasite lysate ±aTc, using α-HA to detect HAD5 and α-Plasmepsin V (PMV) as a loading control. Removal of aTc results in successful knockdown of HAD5. Approximate expected protein masses: PMV, 51 kDa; PfHAD5-3xHA, 32 kDa. C, fold change in parasitemia over time of HAD5^KD parasites grown in varying concentrations of aTc. Data represent mean ± SEM of three independent experiments with technical duplicates. Significance was determined by one-way ANOVA with Fisher's LSD; ∗p = 0.03; ∗∗∗p < 0.001. D, fold change in parasitemia of HAD5^KD parasites grown in varying aTc concentrations with D-mannose rescue. Significance was determined by ordinary two-way ANOVA with Tukey's correction for multiple comparisons. ∗∗∗∗p < 0.0001, ns = not significant. Data represent mean ± SEM of three independent experiments with technical duplicates. E, bright-field images of Giemsa-stained thin-smear synchronized parasites over time. aTc, anhydrotetracycline; HPI, hours postinvasion.

Figure 3

Knockdown of HAD5 disrupts GPI anchor biosynthesis.A, model of the predicted effect that knocking down HAD5 will have on GPI anchor precursor synthesis and subsequent anchoring of GPI-APs. B, representative autoradiography film of GPI anchor precursors from [³H]GlcN-labeled parasites. Bands toward the top migrated farthest on a silica TLC plate, indicating they are less polar and less mannosylated. C, the radiographic signal was quantified and is represented as proportions of total signal. Band numbers indicate the corresponding band from A. Shown are the mean and SEM of three independent experiments, analyzed by ordinary two-way ANOVA with Fisher's LSD test. ∗p = 0.038, ∗∗p = 0.008. D and E, dose–response curve of parasite growth in the presence of GlcN (D) or mannosamine (ManN; E). Data represent the means and SEM of three independent experiments with technical replicates. Aps, anchored proteins; E, ethanolamine; GlcN, glucosamine; GPI, glycosylphosphatidylinositol; Ins, inositol; M, mannose; M1P, mannose 1-phosphate; M6P, mannose 6-phosphate.

Figure 4

Knockdown of HAD5 diminishes membrane anchoring of the egress and invasion protein MSP1.A, representative immunofluorescent images of mechanically freed merozoites that were grown in ±aTc conditions and schizont enriched by E64 treatment. B, quantification of MSP1 signal from A. Data points represent three independent experiments, each with >25 observed merozoites, removing those under a threshold of 10,000 RFU. Bar graphs represent the mean ± SEM of all data points. Statistics were performed by Mann–Whitney test. ∗∗p = 0.008. aTc, anhydrotetracycline; E64, epoxysuccinyl-L-leucylamido(4-guanidino)butane; MSP1, merozoite surface protein 1.

Figure 5

HAD5 is sufficiently distinct from human PMMs to be specifically inhibited.A, dose–response curve of compound D9 against recombinant PfHAD5, HsPMM1, and HsPMM2. Data represent the mean ± SEM of three independent experiments, each with technical replicates. B, activity of PfHAD5 or HsPMM1 was assayed after preincubating enzymes for the given time with 416 μM of D9 prior to adding the preincubation to the reaction mix (final [D9] = 50 μM). As a control (ctrl), enzymes were incubated with an equal volume of water for 60 min. Statistics were performed with an ordinary two-way ANOVA, using Dunnett's test for multiple comparisons. ∗∗p = 0.0046, ∗∗∗∗p < 0.0001, ns, not significant. C, 3.5 Å resolution crystal structure of PfHAD5 (orange) aligned to HsPMM1 (cyan; PDB 2FUC) with Mg²⁺ ions (purple). Indicated are the cap and core domains typical of HAD enzymes. HAD, haloacid dehalogenase; PMM, phosphomannomutase.