Molecular & Biochemical Parasitology 154 (2007) 125–133 Characterization of a serotonin transporter in the parasitic flatworm, Schistosoma mansoni: Cloning, expression and functional analysisଝ Nicholas Patocka, Paula Ribeiro ∗ Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9 Received 7 January 2007; received in revised form 14 March 2007; accepted 19 March 2007 Available online 25 March 2007 Abstract The biogenic amine serotonin (5-hydroxytryptamine: 5HT) is a widely distributed neuroactive substance of vertebrates and invertebrates. Among parasitic flatworms, in particularly the bloodfluke, Schistosoma mansoni, 5HT is an important modulator of neuromuscular function and metabolism. Previous work has shown that schistosomes take up 5HT from host blood via a carrier mediated mechanism. This transport is thought to contribute to the control of schistosome motility in the bloodstream and is essential for survival of the parasite. Here we provide the first molecular evidence for the existence of a 5HT transporter in S. mansoni. A cDNA showing high homology with plasma membrane serotonin transporters (SERT) from other species was cloned and characterized by heterologous expression in cultured HEK293 cells. Functional studies showed that the recombinant 3 schistosome transporter (SmSERT) mediates specific and saturable [ H]-5HT transport with a Kt = 1.30 ± 0.05 ␮M. The heterologously expressed protein was inhibited by classic SERT blockers (clomipramine, fluoxetine, citalopram) and the same drugs also inhibited [3H]-5HT uptake by intact schistosomula in culture, suggesting that SmSERT may be responsible for this transport. Conventional (end-point) and real-time quantitative RT-PCR analyses determined that SmSERT is expressed both in the free-living stage (cercaria) and parasitic forms of S. mansoni but the expression level is significantly higher in the parasites. These results suggest that SmSERT is upregulated following cercarial transformation, possibly to mediate the recruitment of exogenous 5HT from the host. © 2007 Elsevier B.V. All rights reserved. Keywords: Serotonin; Transporter; SERT; Biogenic amines; Neurotransmitter; Schistosoma mansoni 1. Introduction well as host-derived signals that modulate motility. One such signal is serotonin (5-hydroxytryptamine: 5HT), a well known The flatworm Schistosoma mansoni is a blood dwelling neurotransmitter and hormone, which is present at high levels in parasite and one of the major causes of schistosomiasis, a debil- mammalian blood. Studies dating back to the 1970s and 1980s itating disease that afflicts over 200 million people worldwide. have shown that exogenous application of 5HT onto schisto- A free-living larva (cercaria) infects humans by direct pene- somes in culture causes a marked increase in parasite motility tration through the skin and is transformed into an immature [1–3]. The stimulation is due to an increase in the frequency parasitic form (schistosomula) that rapidly enters the circulation of muscle contraction [3,4] and also an increase in carbohy- and begins to develop into sexually mature worms. During this drate metabolism [3,5,6], which makes more energy available development, the young parasite undergoes a complex migra- for movement. The effects on motility are seen in intact ani- tion through the heart and lungs towards the mesenteric veins mals that are not permeabilized, suggesting 5HT is either acting of the hepatic portal system, where the adult worms typically through surface receptors, or is taken internally via a specific reside. This migration is vital to schistosome survival and is transporter and acting directly on the musculature. coordinated by the parasite’s own neuromuscular system, as Plasma membrane serotonin transporters (SERT) were first identified in the mammalian central nervous system (CNS), where they mediate re-uptake of the amine across the presynap- tic membrane. This is part of a mechanism designed to inactivate ଝ Note: The nucleotide sequence reported in this paper has been deposited to GenBank and assigned an accession number of EF061308. 5HT by quickly removing it from the synaptic cleft. SERTs have ∗ Corresponding author. Tel.: +1 514 398 7607; fax: +1 514 398 7857. since been reported in the CNS of other organisms and also non- E-mail address: [email protected] (P. Ribeiro). neuronal tissues that store 5HT [7–9]. Serotonin transporters 0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.03.010 126 N. Patocka, P. Ribeiro / Molecular & Biochemical Parasitology 154 (2007) 125–133 have not yet been cloned from any of the parasitic worms, includ- anchor primer. For 5 RACE, adult S. mansoni total RNA ing S. mansoni. There is, however, good biochemical evidence (3.5 ␮g) was reverse-transcribed using a gene-specific primer to suggest that a SERT-like mechanism of transport is con- (5-CAAATAGCTCACGTCTACCAG-3) and the resulting first served in these organisms. A concentrative, sodium-dependent strand cDNA was modified by the addition of an anchor serotonin transport system was reported in S. mansoni and the sequence to the 5 end, as described in the kit protocol (5 cestode, Hymenolepis diminuta. Moreover, this transport was RACE, Invitrogen). The 5 end tailed cDNA was used as a blocked by treatment with antidepressant drugs, such as flu- template for PCR with a gene-specific reverse primer (5- oxetine (Prozac) [10–14], which are classic SERT inhibitors GAAACACTGTCCATGATTGCTG-3) and a forward primer [15]. Here we report the cloning, functional characterization targeting the anchor sequence at the 5 end. This was fol- and developmental expression analysis of a S. mansoni cDNA lowed by a second round of PCR using an internal gene (SmSERT) that is homologous with previously cloned SERTs specific reverse primer (5-TAACATAAGGCAAAGTAGCAG- from other species. The results provide the first molecular evi- 3) and the same forward anchor primer. 3 and 5 RACE dence for the existence of a 5HT transporter in schistosomes and products were cloned into vector pGEM-T (Promega) and con- further suggest that SmSERT mediates the intake of exogenous firmed by DNA sequencing of two separate clones. Finally, 5HT in vivo. to obtain the full length SmSERT, we designed primers tar- geting the predicted start and stop codons (forward primer: 2. Materials and methods 5-ATGAGTGTATCAAGTCAAAAATTCA-3; reverse primer: 5-TTATAATTTATTTGTTTTGAATG-3) and amplified the 2.1. Parasites cDNA directly from oligo-dT reverse-transcribed total S. man- soni RNA. Once again, products were cloned into pGEM-T and The Puerto Rican strain of S. mansoni was used throughout at least two separate clones were sequenced. these experiments. Infected snails (Biomphalaria glabrata) were obtained from the Biomedical Research Institute (Bethesda, 2.3. Heterologous expression studies MD) and were induced to shed S. mansoni cercaria by expo- sure to artificial light for 1 h at room temperature. Schistosomula For functional expression and pharmacological analyses of were obtained by in vitro transformation of cercaria, using the SmSERT, the full-length cDNA was cloned into the pCI- mechanical tail-shearing method [16]. To obtain adult parasites, Neo vector (Promega) (pCI-neo-SmSERT) and expressed in CD1 female mice were infected with cercaria by active penetra- mammalian cells. HEK293 cells were routinely cultured in tion through the skin. Approximately, 7–8 weeks post-infection Dulbecco’s Modified Essential Media (DMEM) supplemented the mice were sacrificed and the adult worms were collected by with 20 mM HEPES and 10% heat inactivated fetal calf perfusion of the livers and mesenteric veins, as described [16]. serum. For transfection, HEK293 cells were seeded in 24 well plates (1 × 105 cells/well) and allowed to grow overnight. The 2.2. Cloning of SmSERT following day, cells were transiently transfected with pCI-Neo- SmSERT or empty vector for the mock control. Transfections A first analysis of schistosome Expressed Sequence Tags were performed with the use of Fugene 6 (Roche), according (EST) available (www.genedb.org/genedb/smansoni/) identified to the recommendations of the manufacturer. Two days post two overlapping SERT-like ESTs, one belonging to S. man- transfection, cells were washed twice in warm TB (10 mM soni (Sm26427) and the other to the closely related species, Hepes pH 7.5, 150 mM NaCl, 2 mM KCl, 1 mM CaCl2,1mM S. japonicum (BU710860). To amplify these sequences, we MgCl2) and incubated in DMEM (400 ␮l/well) containing extracted total RNA from adult S. mansoni worms using TRIzol 2.22 × 106 DPM/well 5-hydroxy[G-3H]tryptamine trifluroac- (Invitrogen), as per the recommendations of the manufac- etate (98.0 Ci/mmol) and sufficient unlabeled 5HT to produce turer. The RNA was oligo-dT reverse-transcribed, according the desired final concentration. Incubations were carried out to standard procedures, and the resulting cDNA was subse- for 1 h at room temperature, unless indicated otherwise. Fol- quently used in a PCR reaction with sequence-specific primers lowing incubation, cells were washed three times in cold TB targeting the S. mansoni and S. japonicum ESTs. This pro- and lysed by addition of 2% SDS. Incorporated radioactiv- duced a 655 bp partial SERT-like sequence, corresponding ity was then measured using a liquid scintillation counter. roughly to the predicted mid region of the protein’s open read- Nonspecific uptake was assessed by running parallel exper- ing frame. The