Fig 1.
Amino acid alignment of the F. hepatica taurine transporter (Fh TauT) with the human serotonin transporter (Hs SERT), the GABA-transporter-2 of C. sinensis (Cs GAT2), the human GABA-transporter-2 (Hs GAT2) and the human taurine transporter (Hs TauT).
Amino acids are represented in their single-letter code. Invariant sequences are denoted by white letters boxed in red, sequences with conservative substitutions are denoted by red letters. The red and blue arrow heads mark amino acids contributing to the first and second Na+ binding sites, respectively, in the crystal structure of Hs SERT. The green arrow heads highlight residues involved in the coordination of Cl- in SERT. The cysteine residues in the second extracellular loop, which form a putative disulfide bridge, are indicated by magenta arrow heads. The brown arrow heads indicate candidate sites for N-linked glycosylation predicted by NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/).
Fig 2.
Phylogenetic relation of the F. hepatica taurine transporter with transporters of other organisms.
The neighbor-joining method was used to construct the phylogenetic tree by comparing amino acid sequences of transporters (for accession numbers of sequences see materials and methods) with MEGA7 [34]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates, Poisson correction) are shown next to the branches. The scale bar reflects the likelihood that a change in amino acid occurred on any given branch. The transporters written in green indicate those SLC6 family members, where the substrate uptake has been verified in biochemical experiments and/or by electrophysiological recordings. For transporters written in black, the functional assignment is listed as deposited in the data bases; it is apparently based on sequence homology.
Fig 3.
Cellular localization of (A, B) and substrate uptake (C, D) by the F. hepatica (A, C) and the human taurine transporter (B, D) after heterologous expression in HEK293 cells. HEK293 cells were stably transfected with plasmids encoding fluorescently tagged versions of the F. hepatica (YFP-FhTauT, panel A) and the human (CFP-HsTauT, panel B) taurine transporter. The cellular distribution of the tagged transporters was visualized by confocal microscopy (left-hand images). The cell surface was delineated by staining with trypan blue (images in the middle). The captured confocal images were overlaid (right-hand images). C & D: HEK 293 cells (1*105 cells/well) expressing the YFP-tagged F. hepatica taurine transporter (circles in C) and the YFP-tagged human taurine transporter (circles in D) or untransfected HEK 293 cells (open triangles in C) were incubated for 10 min in the presence of the indicated concentration of [3H]taurine. The accumulated radioactivity was determined as described under Materials and Methods. Data are means ± S.D. of at least three independent experiments, which were carried out in duplicate. The solid lines were drawn by fitting the data to the equation of a rectangular hyperbola.
Fig 4.
GABA- (A) and β-alanine-induced (B) inhibition of [3H]taurine uptake by the F. hepatica and the human taurine transporter after heterologous expression in HEK293 cells. Stably transfected HEK 293 cells (1*105 cells/well) expressing the YFP-tagged F. hepatica taurine transporter (circles, YFP-FhTauT) or the YFP-tagged human taurine transporter (triangles, CFP-HsTauT) were incubated for 10 min in the presence of 0.1 μM [3H]taurine and the indicated concentration of GABA (A) and β-alanine (B). The accumulated radioactivity was determined as described under Materials and Methods. Uptake observed in the absence of any inhibitor was set at 100% to normalize for interassay variability. These 100% control values were 0.89 ± 0.05 and 1.102 ± 0.142 pmol.min-1.10−6 cells for the F. hepatica and the human transporter, respectively. Data are means ± S.D. from three independent experiments. The solid lines were drawn by fitting the data to the equation for a monophasic inhibition curve.
Fig 5.
Sodium- (A, B) and chloride-dependence (C, D) of [3H]taurine uptake by the F. hepatica and the human taurine transporter. HEK293 cells (1*105 cells/well) stably expressing the F. hepatica (A, B) or the human taurine transporter (C, D) were incubated for 10 min in the presence of 0.1 μM [3H]taurine and of the indicated concentrations of sodium (A, B) and chloride (C, D). The ionic strength was kept constant by replacing sodium with choline (A, B) or by substituting chloride with acetate (C, D). Data represent means ± S.D. from three independent experiments carried out in duplicate. The solid lines were drawn by fitting the data to the Hill equation.
Fig 6.
Inhibition of [3H]taurine uptake by unlabeled taurine in the absence and presence of GES.
HEK293 cells (1*105 cells/well) stably expressing the F. hepatica (A, B) or the human taurine transporter (C, D) were incubated for 10 min in the presence of 0.1 μM [3H]taurine and of the indicated concentrations of unlabeled taurine and of GES. Data represent means ± S.D. from three independent experiments carried out in duplicate. The solid lines in panels A and C were drawn by fitting the data to a monophasic inhibition curve. In panels, B and D, the data shown in panels A and C, respectively, were replotted in Dixon plots to visualize the non-competitive and the competitive inhibitory action of GES by the intersecting lines (B) and the parallel lines (D), respectively.
Fig 7.
Detection by immunoblotting of the F. hepatica taurine transporter after heterologous expression (A, B) and in lysates prepared from adult flukes (C). A&B: lysates (20 μg/lane) were prepared from untransfected HEK293 cells (lane 1), from HEK293 cells stably expressing the F. hepatica taurine transporter (lane 2) and a YFP-tagged version of the transporter (lane 3). After electrophoretic separation on an SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane, which was probed with an affinity purified rabbit polyclonal antibody raised against the N-terminus (A) and an anti-GFP antibody (B). C: lysates (1 μg) prepared from untransfected HEK293 cells (lane 1) or from HEK293 expressing the untagged (lane 2) and the YFP-tagged F. hepatica taurine transporter (lane 3) were resolved by denaturing gel electrophoresis together with detergent extracts (40 μg) of adult fluke homogenates (lane 4) and of tegumental particulate material (lane 5). After transfer to nitrocellulose membranes, the immunoreactive bands were visualized as described for panels A & B. The immunoblots are representative of four independent experiments.
Fig 8.
Survival of adult F. hepatica in bile acid-containing medium in the presence and absence of taurine.
A: Adult flukes (12 flukes/condition in individual wells) were incubated at 37°C and 5% CO2 in a humidified atmosphere in Hédon Feig medium in the absence (control, closed circles) or presence of 50 μM taurine (closed circle), of 2 mM GES (GES, closed triangle) or of bile acids (closed triangle, 5 mM cholic acid, 1.5 mM deoxycholic acid and 0.5 mM chenodeoxycholic acid). The number of dead flukes was recorded at the indicated time points. B: Adult flukes were incubated at 37°C and 5% CO2 in a humidified atmosphere in Hédon Feig medium in the absence (control) or presence of bile acids (5 mM cholic acid, 1.5 mM deoxycholic acid and 0.5 mM chenodeoxycholic acid). Where indicated, the medium contained, in addition, 50 μM taurine or the combination of 50 μM taurine and 2 mM GES (GES) for 4h. The number of dead flukes was recorded at the end of the incubation period. The data are from three independent experiments with 11 to 12 flukes/condition. Survival in the presence of bile acids and taurine differed significantly from that observed in the sole presence of bile acids (p = 0.0006, Fisher's exact test) and in the presence of the combination of bile acids, taurine and GES (p = 0.0068, Fisher's exact test), but there was no significant difference between survival in the presence of bile acids and of the combination of bile acids, taurine and GES (p = 0.464). Thus, after Bonferroni correction for multiple testing (with α≤0.016), taurine afforded a statistically significant protection against bile acids.