Serotonin signaling in Schistosoma mansoni: a serotonin-activated G protein-coupled receptor controls parasite movement.

Serotonin is an important neuroactive substance in all the parasitic helminths. In Schistosoma mansoni, 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 S. mansoni. 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 S. mansoni. 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 S. mansoni.

Neurotransmitter transporters in schistosomes: structure, function and prospects for drug discovery.

Neurotransmitter transporters (NTTs) play a fundamental role in the control of neurotransmitter signaling and homeostasis. Sodium symporters of the plasma membrane mediate the cellular uptake of neurotransmitter from the synaptic cleft, whereas proton-driven vesicular transporters sequester the neurotransmitter into synaptic vesicles for subsequent release. Together these transporters control how much transmitter is released and how long it remains in the synaptic cleft, thereby regulating the intensity and duration of signaling. NTTs have been the subject of much research in mammals and there is growing interest in their activities among invertebrates as well. In this review we will focus our attention on NTTs of the parasitic flatworm Schistosoma mansoni. Bloodflukes of the genus Schistosoma are the causative agents of human schistosomiasis, a devastating disease that afflicts over 200 million people worldwide. Schistosomes have a well-developed nervous system and a rich diversity of neurotransmitters, including many of the small-molecule ("classical") neurotransmitters that normally employ NTTs in their mechanism of signaling. Recent advances in schistosome genomics have unveiled numerous NTTs in this parasite, some of which have now been cloned and characterized in vitro. Moreover new genetic and pharmacological evidence suggests that NTTs are required for proper control of neuromuscular signaling and movement of the worm. Among these carriers are proteins that have been successfully targeted for drug discovery in other organisms, in particular sodium symporters for biogenic amine neurotransmitters such as serotonin and dopamine. Our goal in this chapter is to review the current status of research on schistosome NTTs, with emphasis on biogenic amine sodium symporters, and to evaluate their potential for anti-schistosomal drug targeting. Through this discussion we hope to draw attention to this important superfamily of parasite proteins and to identify new directions for future research.

The functional role of a serotonin transporter in Schistosoma mansoni elucidated through immunolocalization and RNA interference (RNAi).

Serotonin is an important neurotransmitter in both vertebrates and invertebrates. In the parasitic flatworm, Schistosoma mansoni, serotonin stimulates worm movement and potentiates muscle contraction. A specific serotonin transporter (SmSERT) was previously cloned from S. mansoni and characterized in vitro. Here we conduct a first investigation of the native protein in the worm so as to elucidate the biological role of SmSERT and to assess its drug targeting potential. Confocal immunofluorescence studies using specific antibodies determined that SmSERT is expressed predominantly in the nervous system both in adult worms and larvae (schistosomula). SmSERT immunoreactivity was detected in the main nerve cords of the central nervous system and the peripheral nerve plexus of the body wall in adult males and females, in apparent nerve endings of the male tubercles and possibly the male tegument. In the larvae, SmSERT localized mainly to the peripheral nerve plexus of the body wall. Co-localization experiments showed that the pattern of SmSERT expression coincides with that of serotonin itself, suggesting that SmSERT is present in serotonergic neurons. To test whether SmSERT is involved in the motor effects of serotonin, we treated S. mansoni schistosomula with SmSERT blockers or SmSERT-specific short-interfering RNAs (siRNAs) and then recorded larval motility, using a quantitative imaging assay. In both cases, the treatment produced a strongly hyperactive phenotype, corresponding to a ~3-fold increase in larval motility, roughly the same effect as treatment with an excess of exogenous serotonin. The siRNA effect correlated with a ≈50% decrease in expression of the SmSERT when tested by real-time qPCR. To test if SmSERT mediates transport of exogenous serotonin across the tegument, uptake assays were also performed in intact schistosomula treated with SmSERT siRNAs or an irrelevant siRNA. We found a significant but modest decrease (~25%) in serotonin uptake in the siRNA-suppressed larvae when compared to the negative controls. These results suggest that the SmSERT's function is primarily neuromuscular and may also play a secondary role in the uptake of exogenous (host-derived) serotonin.

The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies.

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. Here, we used a bioinformatics approach to identify the proteasomal constituents of the parasitic trematode Schistosoma mansoni. A detailed search of the S. mansoni genome database identified a total of 31 putative proteasomal subunits, including 17 subunits of the regulatory (19S) complex and 14 predicted catalytic (20S) subunits. A quantitative real-time RT-PCR analysis of subunit expression levels revealed that the S. mansoni proteasome components are differentially expressed among cercaria, schistosomula, and adult worms. In particular, the data suggest that the proteasome may be downregulated during the early stages of schistosomula development and is subsequently upregulated as the parasite matures to the adult stage. To test for biological relevance, we developed a transfection-based RNA interference method to knockdown the expression of the proteasome subunit, SmRPN11/POH1. Transfection of in vitro transformed S. mansoni schistosomula with specific short-interfering RNAs (siRNAs) diminished SmRPN11/POH1 expression nearly 80%, as determined by quantitative RT-PCR analysis, and also decreased parasite viability 78%, whereas no significant effect could be seen after treatment with the same amount of an irrelevant siRNA. These results indicate that the subunit SmRPN11/POH1 is an essential gene in schistosomes and further suggest an important role for the proteasome in parasite development and survival.

Characterization of a serotonin transporter in the parasitic flatworm, Schistosoma mansoni: cloning, expression and functional analysis.

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 schistosome transporter (SmSERT) mediates specific and saturable [(3)H]-5HT transport with a K(t)=1.30+/-0.05 microM. The heterologously expressed protein was inhibited by classic SERT blockers (clomipramine, fluoxetine, citalopram) and the same drugs also inhibited [(3)H]-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.