Serotonin Signaling in <i>Schistosoma mansoni</i>: A Serotonin–Activated G Protein-Coupled Receptor Controls Parasite Movement

<div><p>Serotonin is an important neuroactive substance in all the parasitic helminths. In <i>Schistosoma mansoni</i>, serotonin is strongly myoexcitatory; it potentiates contraction of the body wall muscles and stimulates motor activity. This is considered to be a critical mechanism of motor control in the parasite, but the mode of action of serotonin is poorly understood. Here we provide the first molecular evidence of a functional serotonin receptor (Sm5HTR) in <i>S. mansoni</i>. The schistosome receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to serotonergic type 7 (5HT7) receptors from other species. Functional expression studies in transfected HEK 293 cells showed that Sm5HTR is a specific serotonin receptor and it signals through an increase in intracellular cAMP, consistent with a 5HT7 signaling mechanism. Immunolocalization studies with a specific anti-Sm5HTR antibody revealed that the receptor is abundantly distributed in the worm's nervous system, including the cerebral ganglia and main nerve cords of the central nervous system and the peripheral innervation of the body wall muscles and tegument. RNA interference (RNAi) was performed both in schistosomulae and adult worms to test whether the receptor is required for parasite motility. The RNAi-suppressed adults and larvae were markedly hypoactive compared to the corresponding controls and they were also resistant to exogenous serotonin treatment. These results show that Sm5HTR is at least one of the receptors responsible for the motor effects of serotonin in <i>S. mansoni</i>. The fact that Sm5HTR is expressed in nerve tissue further suggests that serotonin stimulates movement via this receptor by modulating neuronal output to the musculature. Together, the evidence identifies Sm5HTR as an important neuronal protein and a key component of the motor control apparatus in <i>S. mansoni</i>.</p></div

Immunolocalization of Sm5HTR in schistosomulae.

<p><i>In vitro</i> transformed schistosomulae (3–8 days old) were probed with affinity-purified anti-Sm5HTR antibody and visualized by confocal microscopy. (A) Typical schistosomulum showing Sm5HTR (green) immunoreactivity in varicose nerve fibers along the length of the body (left panel). Animals were co-labelled with TRITC-phalloidin to visualize the musculature (middle panel) and the overlay (right panel) shows punctate bright yellow fluorescence, indicating sites of apparent co-localization between Sm5HTR and the outer circular muscles of the body wall (arrows). (B) Schistosomulae were co-labelled with anti-serotonin antibody (green) and anti-Sm5HTR antibody (red). The overlay shows close proximity between the two signals (solid arrows) in the cerebral ganglia (cg) and the main longitudinal nerve cords (nc). Sites of apparent co-localization are marked by open arrows. Sm5HTR immunoreactivity is also prominent in the developing caecum (ce). (C) Schistosomulum co-labelled with anti-serotonin antibody (green) and anti-Sm5HTR antibody (red). Lateral serotonin-containing nerve fibers (solid arrows) can be seen in close proximity to Sm5HTR immunoreactivity in the body wall region of the larva. cg, cerebral ganglia; ce, caecum; nc, nerve cord. (D) Transmission light and corresponding fluorescence image of a typical negative control. No significant fluorescence could be seen in any of the negative controls probed with secondary antibody only or antigen-preadsorbed antibody. Scale bars, 25 µm.</p

Model of serotonin signaling mediated by Sm5HTR in <i>S. mansoni</i>.

<p>Summary of mechanisms by which serotonin can stimulate worm movement. The results suggest that serotonin works through its Sm5HTR receptor to stimulate interneuronal and/or neuromuscular signaling, indirectly increasing contractility of the musculature. Serotonin may also have direct effects on the muscles but it is unclear if those effects are mediated by Sm5HTR or a different receptor. An additional mechanism by which serotonin may control movement is through modulation of sensory neuronal circuits, where Sm5HTR is abundantly expressed. These could play an important role in mediating locomotory responses to host-derived signals, for example chemotactic responses or other types of host-parasite interaction.</p

RNAi in adult worms.

<p>(A) Adult males and females were electroporated with Sm5HTR siRNA or irrelevant control siRNA and motility was recorded approximately 24 hr post-transfection, using an imaging assay. Worm movement was recorded for periods of 2 min in the presence of added serotonin (10⁻⁴ M) and motility data were normalized relative to the irrelevant siRNA control also treated with serotonin. Each data point is the mean and SEM of 15–19 worms obtained from three separate RNAi experiments. Motility in the RNAi – suppressed animals was significantly different from the scrambled siRNA control at p<0.001 (***) or p<0.01 (**). (B) RNAi knockdown was verified at 24 hr post-transfection by reverse transcription (RT) -qPCR analysis. The % remaining expression was determined by the Pfaffl method and was calculated relative to the corresponding male or female control siRNA group. (C) RNAi knockdown was further verified at the protein level by western blot analysis. Worms were harvested 24 hr post-transfection and membrane proteins were extracted. Equal amounts of membrane proteins from test and control worms (mixed males and females) were probed with anti-Sm5HTR antibody or an antibody against a different membrane protein (SmSERT; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003878#ppat.1003878-Patocka2" target="_blank">[25]</a>) as a loading control. Worms electroporated with Sm5HTR siRNAs had significantly less western positive Sm5HTR protein than the control worms electroporated with irrelevant siRNA.</p

RNAi-suppressed larvae are resistant to added serotonin.

<p>RNAi was performed in cultured schistosomulae as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003878#ppat-1003878-g006" target="_blank">Fig. 6</a> except that motility was measured both in the absence and presence of added serotonin (+5HT) at 10⁻⁴ M. Data are the means and SEM of 40 animals and 2–3 transfections per treatment. **, significantly different from basal activity (measured in the absence of serotonin) at p<0.001. ns, not significant at p<0.05.</p

Effects of serotonin agonists and antagonists on larval motility.

<p>Schistosomulae were treated with varying amounts of serotonin (5HT) (A) or o-methyl-serotonin (B). Larval motility was quantified as the frequency of body movements (shortening and elongation) per min of observation, using an imaging assay, as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003878#ppat.1003878-Patocka2" target="_blank">[25]</a>. (C) Larval motility was measured in the presence of 10⁻⁴ M serotonin (5HT) added alone or together with a serotonin antagonist also at 10⁻⁴ M. Motility data are the means and SEM of 3 experiments each with 10–12 animals per treatment. ^a Significantly different from the untreated controls (p<0.05); ^bSignificantly different from 5HT-induced motility (p<0.05). MIAN, mianserin; CPH, cyproheptadine.</p

RNA interference (RNAi) in schistosomulae.

<p>Larvae were treated with 50(mock control) and cultured for 8 days. (A) Motility assays were performed using the same imaging assay described above. The data are the means and SEM of 3 experiments, each performed with at least 20 animals in duplicates. (B) For measurements of RNA expression, total RNA was isolated 8- days post-transfection and oligo-dT reverse-transcribed. Quantitative qPCR was performed with primers targeting Sm5HTR and α-tubulin as a housekeeping gene. Expression was calculated relative to the scrambled control sample, using the comparative Pfaffl method. *** Significantly different from control at p<0.0001; ns, not significant at p<0.05. (C) Representative images of schistosomulae transfected with Sm5HTR siRNA compared to scrambled siRNA or untransfected larvae. Images were obtained at 8 days post-transfection. The controls show the characteristic movement of repeated shortening and elongation, whereas the RNAi-suppressed animals are round in shape and appear unable to elongate the body.</p

Phylogenetic tree of serotonin receptors.

<p>The amino acid sequences of known and predicted serotonin receptors were aligned using the ClustalW method and a Neighbor-Joining Best Tree was constructed from the alignment. The alignment included representative examples of all major classes of serotonergic GPCRs but only three clades are shown for simplicity. These include type 2 serotonin receptors (5HT2), type 7 (5HT7) and structurally related type 1 and type 5 receptors (5HT1/5). The outgroup in the alignment (not shown) was a distantly related rat metabotropic glutamate receptor (NP_058707). The tree was rooted to the outgroup and was tested by bootstrap analysis with 1000 iterations. The length of the branches is proportional to the genetic distance between sequences. Sequences were all obtained from the National Center for Biotechnology Information (NCBI). <i>S. mansoni</i> sequences are designated by their <i>S. mansoni</i> GeneDB “smp”designation number and are marked by arrows. Accession numbers for the remaining sequences are as follows: 5HT2_Drosophila (NP_730859); 5HT2_Lymnae (AAC16969.1), 5HT2(ser-1)_C. elegans (AAC15778); 5HT2B_Xenopus (NP_001082744); 5HT2A_human (NP_000612); 5HT2A_mouse (NP_766400); 5HT2B_human (NP_000858); 5HT2C_human (NP_000859); 5HT5B_mouse (NP_034613.2); 5HT5A_human (NP_076917.1); 5HT1_Helisoma (AAQ95277.1); 5HT1A_Xenopus (NP_001079299.1); 5HT1A_human (NP_000515.2); 5HT1A_chicken (NP_001163999.1); 5HT1D_dog (P11614); 5HT1A_pufferfish (O42385); 5HT1_Aplysia (NP_001191550); 5HT1(ser-4)_C. elegans (NP_497452); 5HT_Haemonchus (AAO45883); 5HT7_Xenopus (NP_001079253.1); 5HT7_human (NP_062873.1); 5HT7_chicken (NP_001167605.1) 5HT7_Helisoma (AAQ84306.1); 5HT7_Meleagris (XP_003206102); 5HT7_zebrafish (XP_690599); 5HT7_Clonorchis (GAA29051); 5HT7_Aedes (AF296125); 5HT7 (ser-7)_C. elegans (NP_741730); 5HT_Dugesia.1 (BAA22404); 5HT_Dugesia.4 (BAA22403); 5HT7_Dugesia (BAI44327.1); 5HT4_chicken (XP_414481.2); 5HT4_human (NP_000861.1); 5HT6_chicken (NP_001166911.1); 5HT6_human (NP_000862.1); 5HT6_mouse (NP_067333.1); ratmGlu.8393487; mGluR1_rat (NP_058707).</p

Functional expression of Sm5HTR in transfected HEK 293 cells.

<p>(A) HEK 293 cells were transfected with a plasmid expressing Sm5HTR-FLAG or empty plasmid (mock control) and probed with anti-FLAG antibody, followed by a FITC-conjugated secondary antibody. Typical <i>in situ</i> immunofluorescence results show expression of FLAG-tagged receptor in the test cells. (B) Schematic of the key signaling mechanisms of serotonin-activated GPCRs. Receptors can signal through changes in intracellular cAMP or Ca²⁺, depending on which G protein (Gs, Gi/o, Gq) is activated. Preliminary experiments showed that serotonin activation of Sm5HTR in HEK 293 cells elevated cAMP but had no effect on cytosolic Ca²⁺ (data not shown). AC, adenylate cyclase; PLCβ, phospholipase C-β; IP₃, inositol trisphosphate; ER, endoplasmic reticulum. (C) Sm5HTR activation causes an increase in intracellular cAMP. HEK 293 cells expressing Sm5HTR were treated with biogenic amines, known serotonergic agonists and antagonists, each at 10⁻⁴ M. Antagonists were tested in the presence of 10⁻⁴ M serotonin. After incubation, the cells were lysed and the lysates were assayed for cAMP. The data were normalized relative to control cells transfected with empty plasmid (mock control). Substances tested included: serotonin (5HT), histamine (HA), acetylcholine (ACh), dopamine (DA), octopamine (OA), tyramine (TA), adrenaline (A), metanephrine (MTN), tryptamine (Trp), o-methyl-serotonin (methyl-5HT), buspirone (BUS), 8-Hydroxy-DPAT (DPAT), mianserin (MIAN), chlorpromazine (CHP), cyproheptadine (CPH). ^a significantly different from untreated at p<0.01; ^b significantly different from serotonin –induced cAMP level at p<0.01. Experiments were repeated with variable concentrations of serotonin (D) and o-methyl-serotonin (E) to obtain dose-response curves. Data were baseline-subtracted and normalized relative to the maximum agonist-induced response. Each data point is the mean and SEM of at least three independent experiments performed in duplicates or triplicates.</p

Effects of dopamine and related substances on schistosome motility.

<p>(A) <i>In vitro</i> transformed 3-day-old schistosomula were incubated with test drug, dopamine (DA) or epinine (EPN), each at (10⁻⁴ M) or vehicle (CT, control). Animals were treated for 5 min at room temperature, after which they were examined with a compound microscope equipped with a digital video camera and SimplePCI (Compix Inc.) for image acquisition. Images were recorded for 1 minute (∼3 frames/second) and an estimate of body length in µm was obtained for each animal in every frame. Each tracing shown is of an individual animal and is representative of 12–15 larvae per experiment and 3–4 independent experiments per treatment. (B) Experiments were repeated with various concentrations of test agonist in a range of 10⁻⁷ M–10⁻⁴ M, or in the absence of test substance (CT, control). Images were recorded as above and body length was measured for each frame. Motility is defined as the frequency of length changes (shortening and elongation) per minute of observation, as described in the Methods. The data are presented as the means and SEM of three separate experiments each with 12–15 animals. (C) Schistosomula were treated with test substances at a single concentration or in the absence of drug (CT, control) and motility was measured as above. Dopamine (DA), epinine (EPN), flupenthixol (FLPX), promethazine (PRMZ) were each tested at 50 µM. The remaining substances, adenaline (A), metanephrine (MTN) and haloperidol (HLRD) were tested at 500 µM. The data are the means and SEM of three separate experiments each with 12–15 animals. * Significantly different from the no drug control at P<0.05.</p