The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

Abstract

The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions.

Keywords: 14-3-3 protein; FAD-dependent glycerol-3-phoshate dehydrogenase; Giardia duodenalis; NBDHEX; encystation; energy metabolism; mitosome; nitroreduction.

FIGURE 1

Expression and localization of the glycerol-3-phosphate dehydrogenase (gG3PD) during the differentiation stages of Giardia duodenalis. (A) Western blot from three independent analysis of Triton lysates (20 μg) from G. duodenalis WB-C6 trophozoites (T) and parasites harvested at 6, 12, and 24 h after encystation induction. Immunoblotting was performed with: anti-gG3PD polyclonal serum (α-gG3PD); anti-phosphoacetylglucosamine mutase (α-PGM) (Lopez et al., 2003), to follow the progression of encystation; the anti-α-tubulin (α-α-TUB), as loading control. Molecular size markers (kDa) are reported on the left. The analysis is representative of three independent experiments. (B) Confocal laser scanning microscopy (CLSM) observations of fixed and permeabilized G. duodenalis WB-C6 parasites at different stages: trophozoite (panel a, Trophozoite), encysting parasite after 12 h of encystation (panel b and c, Encystation) and cyst (panel d, Cyst) stained with the mouse polyclonal serum α-gG3PD (red) and the α-g14-3-3 rabbit polyclonal (green). Cyst wall and encystation specific vesicles (ESVs) were stained with Cy3-conjugated α-CWP mAb (gray). Nuclei (N) were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Displayed micrographs correspond to a single z-stack: a and b, ventral stacks; c and d, central stacks encompassing the nuclei. T, transmission light acquisition. Scale bars, 5 μm. Arrows indicate the ventrolateral flanges (VLF). A magnification (zoom) of the indicated area in the merged image is shown. (C) CLSM observation as in (B). Parasites were stained with α-gG3PD (red) and α-Tom40 antiserum (green; Dagley et al., 2009). Nuclei (N) were stained with DAPI (blue). A magnification (zoom) of the indicated area in the merged image is shown. Mitosomes are indicated (m). Images in (B) and (C) are representative of >50 fields analyzed in two independent experiments.

FIGURE 2

Expression of the recombinant FLAG-HA-gG3PD in G. duodenalis and its interaction with g14-3-3. (A) Western blot analysis of Triton lysates from trophozoites (T) or parasites after 12 h of encystation (E) obtained from FLAG-HA-gG3PD transfected line or from the control WBC6 strain. Twenty-microgram of protein extracts were loaded in each lane and separated on a 4–12% SDS-PAGE, then transferred on nitrocellulose membrane and finally probed with the indicated antibodies. Equal protein loading was monitored by anti-TUB Ab, whereas encystation induction was confirmed by anti-gPGM Ab. Molecular size markers (kDa) are reported in the left. (B) Subcellular distribution of gG3PD. Thirty-microgram of fractionated protein lysate, soluble (S) or octyl β-D-glucopyranoside solubilized membrane proteins (M), from trophozoites (T) or 12 h encysting parasites (E) of FLAG-HA-gG3PD transfected parasites or control WB-C6, were treated as described in (A) and probed with the indicated antibodies. Molecular size markers are on the left. (C) Co-immunoprecipitation assay of endogenous g14-3-3 with FLAG-HA-gG3PD. An aliquot (1:5) of the FLAG peptide-eluted material from WB-C6 or FLAG-HA-gG3PD transgenic line, deriving from trophozoites (T) or encysting parasite (E), was separated on 4-12% SDS-PAGE and immunoblotted with anti-HA (α-HA) or anti-g14-3-3 polyclonal serum (α-g14-3-3). Molecular size markers are on the left. Immunoblots of (A–C) are representative of three independent experiments.

FIGURE 3

Expression and intracellular localization of the FLAG-HA-tagged gG3PD deletion mutants in G. duodenalis parasites. (A) Representative western blot analysis of soluble protein lysate (20 μg) from control WB-C6 and transgenic trophozoites (T) or parasites after 12 h of encystation (E), expressing the full-length FLAG-HA-G3PD, or the FLAG-HA-gG3PD_N or _C. Lysates were separated on 4–12% SDS-PAGE and immunoblotted with the indicated antibodies. Arrows on the right indicate the molecular size of the corresponding proteins. Molecular size markers are on the left. The vertical line in the panel corresponding to the α-HA blotting indicates two different times of exposure between the samples of the same gel. The immunoblots are representative of three independent experiments. (B) CLSM observations of fixed and permeabilized transgenic G. duodenalis parasite expressing the FLAG-HA-gG3PD_N or the FLAG-HA-gG3PD_C at different stages: trophozoite (panels a and d, Trophozoite), encysting parasites after 12 h of encystation (panels b and e, Encystation), and cysts (panels c and f, Cyst). Parasites were stained with mouse α-HA mAb (green) and rabbit polyclonal α-g14-3-3 (red). Cyst wall and encystation specific vesicles (ESVs) were stained by Cy3-conjugated α-CWP mAb (gray). Nuclei were DAPI-stained (blue). Displayed micrographs correspond to a single z-stack. T, transmission light acquisition. Scale bars are reported. Arrows indicate the ventral plasma membrane and VLF. Images are representative of >50 fields analyzed in two independent experiments.

FIGURE 4

Evaluation of the gG3PD enzymatic activity. (A) Spectrophotometric analysis of purified HIS-gG3PD. The UV-visible spectrum of HIS-gG3PD (10 mg/ml) in 67 mM of potassium phosphate buffer, pH 7.5, was recorded at 25°C. The insert shows a magnification of the HIS-gG3PD spectrum (solid line) in comparison with the spectrum of authentic FAD (dotted line) recorded in the same buffer. Peak maxima are reported. Spectra are representative of three independent experiments. (B) Assessment of HIS-gG3PD dimerization in vitro. Purified recombinant proteins (3 μmol) were separated on 3–12% Blue Native-PAGE and silver-stained or transferred on polyvinylidene difluoride (PVDF) membrane and probed with anti-HIS mAb. Native size markers (kDa) are indicated on the left. Asterisks indicate HIS-gG3PD monomer (^∗) or dimer (^∗∗). Empty dots indicate HIS-gG3PD_C monomer (°) or dimer (^°°). The arrow indicates HIS-gG3PD_N monomer (<). Native-PAGE and immunoblot are representative of three independent experiments. (C) The enzymatic activities of both the purified HIS-gG3PD and the deletion mutant HIS-gG3PD_N were measured in vitro, by MTT assay, in the presence (+FAD) or absence (no FAD) of 10 μM FAD. The G3PD activity (mean ± SD) from three independent experiments is expressed as nmol of reduced MTT per min per mg of recombinant protein. Statistical analyses were performed using unpaired t-test between the full length and the deletion mutant: ^∗P < 0.05 and ^∗∗P < 0.01. (D) The FAD-glycerol-3-phosphate dehydrogenase activity was measured in protein extract (100 μg) from G. duodenalis trophozoites (Trophozoite) or in parasite after 6 or 12 h from encystation induction (Encystation). The relative enzymatic activity (mean ± SD) from three independent experiments is expressed as the percentage change respect to the value measured in trophozoites. Statistical analyses were performed using unpaired t-test [Trophozoite vs. Encystation 6 h (^∗∗P < 0.01) and Trophozoite vs. Encystation 12 h (ns)], and one-way ANOVA (^°°P < 0.001). (E) Western blot analysis of protein extracts (20 μg) used to assay the gG3PD enzymatic activity (as described in D), extracts were separated on 4–12% SDS-PAGE and immunoblotted with the indicated antibodies. Molecular size markers are indicated on the left. Immunoblot is representative of three independent experiments. (F) The intracellular glycerol content was measured in supernatant from G. duodenalis trophozoites (Trophozoite) and parasites after 6 or 12 h from encystation induction (Encystation). The relative glycerol amount of three independent experiments (mean ± SD) is expressed as the percentage change respect to the amount detected in trophozoites. Statistical analyses were performed using unpaired t-test between Trophozite and Encystation 6 h (^∗∗∗P < 0.001) and Trophozite vs Encystation 12 h (^∗∗P < 0.01). One-way ANOVA confirmed that differences among groups were statistically significant (^°°P < 0.001).

FIGURE 5

Evaluation of NBDHEX effects on G. duodenalis growth and gG3PD enzymatic activity. (A) Survival of G. duodenalis WBC6 trophozoites was determined by methylene blue colorimetric assay after 48 h of treatment with different concentrations, ranging from 0.05 to 10 μM, of NBDHEX (empty dots) or metronidazole (MTZ, empty squares) in microaerophilic growth conditions. Data (mean percentage ± SD) represent three independent experiments, each done in triplicate. The structure of the NBDHEX compound is reported in the insert. (B) The FAD-glycerol-3-phosphate dehydrogenase activity was measured in protein extract (100 μg) from G. duodenalis trophozoites treated for the indicated times with 50 μM NBDHEX or for 6 h with ethanol (Control). The enzymatic activity of three independent experiments (mean ± SD) is expressed as the percentage change respect to the control. Unpaired t-test was performed between control and each time point (^∗P < 0.05 and ^∗∗P < 0.01). One-way ANOVA indicated statistically significant differences among all stages (^°°∘P < 0.0001). (C) Twenty-microgram of protein extracts derived from trophozoites treated as described in (B), were separated on 4–12% SDS-PAGE and immunoblotted. A representative western blot analysis is shown and the antibodies indicated. Molecular size markers are reported in the left. Table in the bottom reports the densitometric analysis of three independent experiments (mean ± SD). Statistical analyses using t-test were not significant.

FIGURE 6

Interaction of NBDHEX with gG3PD. (A) CLSM analysis of fixed and permeabilized G. duodenalis WBC6 trophozoites after 2 h incubation with 50 μM NBDHEX. NBDHEX (green) was directly visualized using the laser light at 488 nm ex. Parasites were stained with rabbit polyclonal anti-g14-3-3 and AlexaFluor 647-conjugated anti-rabbit (red). Nuclei were stained with DAPI (blue). Displayed micrographs correspond to a single z-stack. T, transmission light acquisition. Scale bar is reported. Images are representative of >50 fields analyzed in two independent experiments. (B) Histograms represent the FAD-glycerol-3-phosphate dehydrogenase activity (mean percentage ± SD) of recombinant HIS-gG3PD (16 pmol) purified from HIS-gG3PD-overproducing E. coli after 2 h incubation with 50 μM NBDHEX (NBDHEX) or ethanol (Control). The relative change in the enzymatic activity is given as percentage in relation to the control. Statistical analyses were performed using unpaired t-test (^∗∗∗∗P < 0.0001) on three independent experiments. (C) SDS-PAGE (4–12%) of 2 μg of purified NBDHEX-treated (NBDHEX) or ethanol-treated (Control) HIS-gG3PD, as described in (B), under reducing (+DTT) or not reducing condition. On the left, gel stained with Coomassie blue; on the right, the same gel photographed under UV light prior to staining. Molecular size markers are indicated on the left. (D) MS/MS spectra matching the gG3PD peptides (residues 539–551, left, and residues 859–872, right) carrying a mass increase of 265 Da on the cysteine residues C545 and C865, respectively. The mass shift is compatible with an NBDHEX-derived adduct in which the nitro group was completely reduced to an amine. Data shown in (C) and (D) are representative of three independent experiments.

FIGURE 7

NBDHEX is substrate of HIS-gG3PD. (A) Spectrophotometric analysis of the reaction between NBDHEX and gG3PD. The UV-visible spectrum of NBDHEX (100 μM) in 67 mM potassium phosphate buffer (pH 7.5) was recorded at 25°C either in the presence of 17 mM g3p (red line, +g3p) or 80 nM HIS-gG3PD (dashed black line, +HIS-gG3PD). The NBDHEX (100 μM) UV-visible spectrum was also recorded at different time points (0, 10, 20, 40, and 80 min) in the presence of both 17 mM g3p and 80 nM HIS-gG3PD (+HIS-gG3PD+g3p). Arrows indicate the wavelength (nm) of the maximal absorption. (B) Fluorescence spectra of 100 μM NBDHEX (excitation at 430 nm) after 80 min of incubation with 80 nM HIS-gG3PD, either in the presence (solid line) or absence (dashed line) of 17 mM g3p. Fluorimetric analyses were recorded at 25°C in 67 mM potassium phosphate buffer (pH 7.5). NBDHEX fluorescence emission has a maximum peak wavelength at 525 nm. Fluorescence intensity (y axis) is arbitrary. The change in color of the two reactions is shown in the insert. (C) The UV-visible spectrum of NBDHEX (100 μM) in 67 mM potassium phosphate buffer (pH 7.5) was recorded at 25°C before (solid line) and after (dashed line) treatment with sodium dithionite (Na₂S₂O₄). The wavelengths (nm) of the maximal absorption are indicated with arrows. (D) Fluorescence spectra of 100 μM NBDHEX (excitation at 430 nm) before (solid line) and after Na₂S₂O₄ (dashed line). The slight change in color of the two conditions is shown in the insert. Spectra and images from (A–D) are representative of three independent experiments. (E) SDS-PAGE (4–12%) of 2 μg of HIS-gG3PD after 80 min of incubation at 25°C with NBDHEX (+NBDHEX) in the presence or not of g3p, or with ethanol (no NBDHEX). Gel was photographed under UV light (UV, right) before staining with Coomassie blue (Coomassie, left). Molecular size markers are reported in the left. Images are representative of three independent experiments. (F) Comparison of the UV-visible spectra of NBDHEX (green and orange line) or MTZ (solid and dashed line) incubated with HIS-gG3PD and g3p (G3PD) at time 0 min and after 80 min of incubation at 25°C (T0 and T80, respectively). Arrows indicate the wavelengths (nm) of the maximal absorption. Spectra are representative of three independent experiments.