The nucloetide sequence (accession number DF144738.1) of the putative high-affinity choline transporter of C. sinensis (CsChT) was acquired through a search on the National Center for Biotechnology Information (NCBI). The corresponding amino acid sequence encodes the solute carrier family 5 member 7 (high-affinity choline transporter, SLC5A7; accession number: GAA57421.1). Other high-affinity choline transporter genes from flukes were obtained from the NCBI database, and the sequences were aligned using the Clustal W program (http://clustalw.ddbj.nig.ac.jp). The evolutionary history of the choline transporter in flukes was inferred using MEGA11 software (http://www.megasoftware.net) by implementing maximum-likelihood analysis with a bootstrap test of 1,000 pseudo replications to enhance robustness.

The topology of membrane spanning was determined using the TMpred server (https://ftp.vital-it.ch/pub/software/unix/tmpred/www/TMPRED_form.html), which predicted the presence of 13 potential transmembrane domains. The tertiary structure of CsChT was further analyzed by submitting the putative CsChT sequences to the SWISS-MODEL server (https://swissmodel.expasy.org), a homology-base modeling tool that identified suitable structure templates as described elsewhere [21]. We then implemented Ramachandran plots and ERRAT to assess the quality of the tertiary structure model and identify potential errors. The generated model was refined to improve accuracy using a combination of 2 programs, Mode Refiner and Galaxy Refine. Finally, the final predicted structure was selected and visualized using UCSF CHIMERA software.

To obtain adult C. sinensis worms, we collected specimens of Pseudorasbora parva and Gnathopogon coreanus from the Jinju Nam River, an area in Korea that is highly associated with metacercariae. The fishes were digested enzymatically using an artificial digestive solution consisting of 0.6% pepsin and 0.7% HCl at pH 2.0. The digestion process was carried out for 6 h in a shaking water bath at 37°C with gentle agitation. Undigested material were removed by filtration using a five-fold gauze. The residue was washed 5 times with a normal saline solution (0.85% NaCl) to eliminate the digestive solution. Metacercariae were then isolated by filtering the mixture through a 200-μm stainless wire mesh, resulting in the collection of approximately 500 metacercariae. Experimental infections were carried out using 5–6-week-old male FVB/NJ mice (Central Lab Animal Inc., Seoul, Korea), which were orally infected with 30 metacercariae. Six weeks after infection, the mice feces were examined for the presence of eggs using the formalin-ether sedimentation method [22]. Finally, adult C. sinensis worms were collected from the liver margin and bile ducts approximately 8 weeks after the initial infection. The animal experiment protocol was reviewed and approved by the institutional animal care and use committee of Inha University (approval no. INHA161208-460), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The mice were cared for at the Inha University Animal Facility following the National Animal Care Policies (Accredited Unit, Korea FDA; unit number 36).

A total of 500 ng of RNA extracted from adult worm was used to synthesize cDNA via reverse transcription. The process used 5 units of Avian Myeloblastosis Virus reverse transcriptase XL kit (Takara, Kusatsu, Japan), 10 mM of dNTP, and 2.5 μM of oligo dT primers in a total volume of 30 μl. The mixture was then incubated at 42°C for 30 min, followed by a temperature increase to 99°C for 5 min. The mixture was then held at 4°C.

The full length of the putative CsChT gene was generated using overlap extension PCR, which involved the designing of 3 sets of primers to generate 3 gene segments, each with overlapping regions of at least 20 base pairs. The reaction mixture for each segment consisted of 3 μl of cDNA, 3 μl of dNTP (2.5 mM each), 1 unit of Ex Taq DNA polymerase (Takara), 3 μl of 10×PCR reaction buffer, 1 μl of forward and reverse primer (10 μM) each (Supplementary Table S1), and DNase/RNase-free water to adjust the final volume to 30 μl. The thermocycler conditions were as follows: predenaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 45 sec, and a final extension at 72°C for 5 min. The amplicon was purified using a Gel Extraction Kit (GeneAll Biotechnology, Seoul, Korea) and subcloned into a pGEM-T easy TA cloning vector (Promega, Madison, WI, USA) for sequence confirmation. After verifying the sequence (Macrogen, Seoul, Korea), the plasmid DNA was digested with EcoRI to obtain the overlapping PCR templates (Supplementary Fig. S1A). The reaction mixture for overlapping PCR consisted of 1 μl of each PCR segment, 3 μl of dNTPs (2.5 mM each), 2 units of LA taq DNA polymerase (Takara), 3 μl of 10×PCR reaction buffer, 2 μl of 25 mM MgCl₂, 1 μl of first forward and third reverse primers (10 μM) each, and DNase/RNase-free water to adjust the final volume to 30 μl. The overlapping PCR conditions were as follows: predenaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 5 min (Supplementary Fig. S1B).

To measure choline uptake properties, we employed the Xenopus laevis oocyte expression system, which has been widely used to characterize transporters, as described previously [23]. Capped cRNA was synthesized in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (ThermoFisher Scientific, Carisbad, MA, USA) using cDNA linearized with the Xho I (Enzynomics, Daejeon, Korea) restriction enzyme. Fifty nanoliters of cRNA was injected into defolliculated oocytes at a rate of 50 ng per oocyte, while the control group was injected with an equal volume of distilled water. The injected oocytes were then incubated at 18°C in Barth’s solution (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.4 mM CaCl₂, 0.8 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM HEPES, pH 7.4) supplemented with 50 μg/ml gentamicin and 2 mM of sodium pyruvate. After an incubation period of 2 to 3 days, CsChT expression was assessed by measuring the uptake of [³H] choline (PerkinElmer, Waltham, MA, USA) at 22–25°C in ND96 solution (96 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.4) for 1 h. The uptake reaction was stopped by the addition of ice-cold ND96 solution, and the oocytes were subsequently washed 4 times with the same solution to remove residual substrate. The oocytes were dissolved with 10% SDS solution under vigorous shaking for 1 h, followed by the addition of 2 ml of the scintillation cocktail, Ultima Gold AB (PerkinElmer). The radioactivity of dissolved oocytes was measured using a MicroBeta² β-Counter (PerkinElmer).

The trans-stimulatory effect of CsChT on the efflux of radiolabeled choline was investigated by preloading CsChT-expressing oocytes with [³H] choline (10 μM in the medium) for 90 min. The oocytes were then transferred to medium that was either contained excess amount of (100 μM and 1,000 μM each) or lacked unlabeled choline and incubated for 1 h [24].

The kinetic parameters associated with the CsChT-mediated uptake of choline were estimated using the following equation: v=Vmax×S/(Km+S), where v represents the substrate uptake rate (pmol/h/oocyte), S denotes the substrate concentration in the medium (μM), Km represents the Michaelis–Menten constant (μM), and Vmax represents the maximum uptake rate (pmol/h/oocyte). To determine these kinetic parameters, the equation was fitted to the choline transport velocity, which was obtained by subtracting the transport velocity of choline in noninjected oocytes from that in CsChT-expressing oocytes. This fitting graph was constructed using an iterative nonlinear least squares method using the MULTI program [25]. The input data, consisting of weighted values using the reciprocal of the observed values, and then fitted using the Damping Gauss Newton method [25]. Subsequently, the fitted line was transformed into the 1/S versus 1/V format for Lineweaver–Burk analysis.

The full length of CsChT (GAA57421) is composed of 1,710 base pairs, which encodes 569 amino acid residues (aa.) within an open reading frame that encodes a protein weighing 62.7 kDa. One to three amino acids of the N-terminus tail is positioned in the extracellular region and the region is predicted to contain 13 putative transmembrane segments. The C-terminus tail spans amino acid residues 498–569 and is located within the intracellular environment (Fig. 1A; Supplementary Fig. S2).

To determine the tertiary structure of CsChT using the SWISS-MODEL server, we found the human sodium/glucose cotransporter 2 (hSGLT2, PDB ID: 7vsi.1.A) to be an excellent template for structural fitting. Analysis through a Ramachandran plot indicated that 90.1% and 0.2% of the model occupied favorable and disallowed regions, respectively. The initial ab initio model structure had a quality verification score of 87.6%, according to ERRAT results. Model refinement significantly improved the 3-dimensional structure, resulting in 95.4% and 0.2% of the model occupying favorable and disallowed regions, respectively, based on the Ramachandran plot. ERRAT analysis of the refined model indicated a final allowed CsChT structure quality of 90.3% (Fig. 1B). The final tertiary structure, consisting of amino acid residues 3 to 502 and encompassing 13 α-helical transmembrane domains, is shown in Fig. 1B.

The relationships between putative choline transporter-related genes were determined by comparing the CsChT sequence to those of high-affinity-related genes from other flukes, which were obtained from the NCBI database. Sequence analysis indicates the following levels of homology among choline transporter-related proteins of other flukes to CsChT: Fasciola gigantica (TPP67279.1), 71%; Fasciola hepatica (THD26485.1), 72%; Opisthorchis felineus (TGZ71349.1), 97%; Paragonimus westermani (KAF8572153.1), 76%; Schistosoma bovis (RTG82711.1), 70%; Schistosoma haematobium (XP_035586184.1), 69%; Schistosoma japonicum (TNN15232.1), 72%; and Schistosoma mansoni (XP_018652611.1), 63% (Fig. 1C; Table 1).

The transport properties of various substrates via CsChT were investigated using the Xenopus oocyte expression system. Specifically, we examined the uptake rates of isotope-labeled choline (for choline transporter), arginine (an amino acid), α-ketoglutarate (a dicarboxylate), p-aminohippurate (an organic anion), taurocholate (a bile acid), and estrone sulfate (a steroid hormone). The results reveal that the uptake rate of [³H] choline in oocytes expressing CsChT was approximately 4.5 times higher than that of the control group (control: 9.6± 1.2 pmol/oocyte/h, CsChT: 43.6±3.6 pmol/oocyte/h). In contrast, we observed no significant uptake of amino acids, organic anions, dicarboxylates, steroid hormones, and bile acids (Fig. 2).

The CsChT-mediated uptake of [³H] choline was dependent on both expression time (i.e., increasing for 1–3 days, Fig. 3A) and incubation time (i.e., uptake intensified between 15 to 75 min, Fig. 3B). Similarly, choline uptake was dependent on sodium ions, which we confirmed by replacing the extracellular sodium content (ND96, containing 96 mM Na⁺ ion) with equimolar amounts of LiCl and NMDG (N-methyl-D-glucosamine). Notably, CsChT-mediated choline uptake was significantly diminished during one hour in the presence of LiCl and NMDG (Fig. 4A). We searched for putative sodium ion binding residues within the conserved domain database of NCBI, and we found a typical solute-binding domain within CsChT that is homologous to that of Na⁺- and Cl⁻-dependent choline cotransporters. We identified several sodium ion binding residues (Ala 59, Val 62, Ala 338, Ser 341, and Ser 342) within the conserved domain of CsChT, demonstrating that these residues are highly conserved across diverse organisms (Fig. 4B). These residues are located within the second and ninth transmembrane cores, with 2 serine residues positioned in the intracellular region (Fig. 1B). The spatial arrangement of these sodium-binding residues creates a pocket that is conducive to sodium ion binding, which is essential for transporting choline (Fig. 4B).

We examined the transstimulatory property of choline transport via CsChT by adding choline to the medium at concentrations of 100 μM and 1,000 μM, which are 10 and 100 times higher than preloaded choline concentration, respectively. We did not observe any significant CsChT-mediated efflux of preloaded isotope-labeled choline (Fig. 5A). The uptake of [³H] choline via CsChT was concentration-dependent, displaying saturable kinetics that followed the Michaelis–Menten equation (Fig. 5B). Lineweaver–Burk analysis of choline uptake yielded a Km value of 8.3 μM and a Vmax value of 61.0 pmol/oocyte/h (Fig. 5B).