Amira Taman

Investigation of a dopamine receptor in Schistosoma mansoni: Functional studies and immunolocalization

A dopamine receptor (SmD2) was cloned from adult Schistosoma mansoni. The receptor has the classical heptahelical topology of class A (rhodopsin-like) G protein-coupled receptors (GPCR) and shares sequence homology with D2-like receptors from other species. The full length SmD2 cDNA was expressed in the yeast Saccharomyces cerevisiae and mammalian HEK293 cells. Functional assays in both expression systems revealed that SmD2 was responsive to dopamine in a dose-dependent manner, whereas other structurally related amines had no effect. Activation of SmD2 in mammalian cells caused an elevation in intracellular cAMP but not calcium, suggesting that the receptor coupled to Gs and the stimulation of adenylate cyclase. Pharmacological studies showed that the S. mansoni dopamine receptor was inhibited by apomorphine, a classical dopamine agonist, as well as known dopaminergic antagonists, including chlorpromazine, spiperone and haloperidol. SmD2 immunoreactivity was detected in membrane protein fractions of S. mansoni cercaria, in vitro transformed schistosomula and adult parasites, using a specific peptide antibody. When tested by confocal immunofluorescence, SmD2 was detected in the subtegumental somatic musculature and acetabulum of all larval stages tested. In the adults, SmD2 was enriched in the somatic muscles and, to a lesser extent, the muscular lining of the caecum. The results suggest that SmD2 is an important component of the neuromuscular system in schistosomes.

Glutamate-mediated signaling in Schistosoma mansoni: a novel glutamate receptor is expressed in neurons and the female reproductive tract.

l-Glutamate is a major neurotransmitter of both vertebrates and invertebrates. Earlier studies have shown that glutamate stimulates neuromuscular activity in the bloodfluke, Schistosoma mansoni, but its mode of action is unknown. Here we describe a novel glutamate receptor in S. mansoni (SmGluR), the first of its kind to be identified in a parasitic flatworm. SmGluR belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to metabotropic glutamate receptors from other species. The full-length receptor cDNA was cloned, stably expressed in HEK-293 cells and shown to be activated by glutamate, whereas aspartate and the glutamate derivative, gamma-aminobutyric acid (GABA) had no significant effect. Among the classical (mammalian) agonists and antagonists tested, only LY341495 was able to interact with the schistosome receptor, suggesting that the pharmacological profile of SmGluR is substantially different from that of receptors in the host. The presence of SmGluR in the parasite was verified by immunoprecipitation and Western blot analyses, using a specific peptide antibody. Confocal immunolocalization studies revealed that SmGluR is strongly expressed in the nervous system of adult worms and larvae. In the adults, the receptor was detected in the longitudinal nerve cords and cerebral commissures, as well as the peripheral nerve fibers and plexuses innervating the acetabulum and the somatic musculature. Outside the nervous system, SmGluR was detected along the length of the female reproductive system, including the oviduct, ootype and the uterus. A comparative expression analysis at the RNA level revealed that SmGluR is expressed at about the same level in cercaria and adult stages, as determined by quantitative reverse-transcription PCR. The results identify SmGluR as an important neuronal receptor and provide the first molecular evidence for a glutamate signaling system in schistosomes.

A Novel G Protein-Coupled Receptor of Schistosoma mansoni (SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of “orphan” amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.

Present-day anthelmintics and perspectives on future new targets.

In absence of vaccines for the majority of helminths, chemotherapy is still the mainstay for controlling human helminthiases. However, a limited number of drugs are available in the market to combat parasitic helminths in human. Besides, the development and spread of drug resistance have declined the use of most currently available anthelmintics. Clearly, availability of new anthelmintic agents will be essential in the next few years. More research into the mechanisms of drug actions and their targets are eminent for the discovery and development of novel anthelmintic agents. Recent drug discovery techniques mostly rely on mechanism-based screening of compounds on heterologously expressed targets in bacterial, mammalian or yeast cells. Although this is usually a successful approach, it is money- and time-consuming; meanwhile, pharmaceutical companies prefer the tested target that is chosen based on basic research. The nervous system is the site of action of several chemotherapeutics including pesticides and antinematode drugs; accordingly, the nervous system continues to be a promising target. Recent advances in exploring helminths’ nervous system, neurotransmitters and receptors have paved the way for the development of potential agents targeting the nervous system and its components.

First insight into the effect of single oral dose therapy with artemisinin-naphthoquine phosphate combination in a mouse model of Schistosoma mansoni infection.

Praziquantel is the current drug of choice against schistosomiasis. The dependency on praziquantel exclusively is problematic, given the spread of the disease and the threat of drug resistance. This study investigates an alternative antischistosomal drug using the compound naphthoquine phosphate tablet, which is a novel single oral dose antimalarial drug, containing a combination of naphthoquine phosphate and artemisinin. In the present study, the therapeutic efficacies of different artemisinin-naphthoquine phosphate combination-dosing protocols were evaluated in experimentally infected mice harbouring juvenile or adult stages of Schistosoma mansoni (Egyptian strain). The study shows that the oral administration of artemisinin-naphthoquine phosphate combination in a single dose of 400 mg/kg on day 7 p.i. resulted in a significant worm burden reduction of 95.07%. When used at a dose of 600 mg/kg on day 21 p.i., all female worms were killed before depositing eggs, resulting in complete absence of eggs in hepatic and intestinal tissues. The same dose given on day 42 p.i. reduced total and female worm burdens by 93.36% and 94.17%, respectively. In addition, artemisinin-naphthoquine phosphate combination induced significant reductions of 80.18% and 76.73% in the hepatic and intestinal tissue egg loads, respectively. Artemisinin-naphthoquine phosphate combination also induced significant alterations in the oogram pattern with elevated levels of dead eggs. Antipathological activities were evident in the amelioration of hepatic granulomata. Our findings hold promise for the development of a novel antischistosomal drug using an artemisinin-naphthoquine phosphate combination. Further in vitro and in vivo studies should be launched to elucidate the possible mechanism/s of action and to study the effect of artemisinin-naphthoquine phosphate combination on other human schistosomes.

Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3′end, suggesting the transcript is full-length. The cloned cDNA was subsequently expressed in bacteria and shown to encode a functional glutamate-binding protein. Other studies, using a specific peptide antibody, determined that SmGBP exists in two forms, a monomer of the expected size and a stable but non-covalent dimer. The monomer and dimer are both present in the membrane fraction of S. mansoni and are resistant to extraction with high-salt, alkaline pH and urea, suggesting SmGBP is either an integral membrane protein or a peripheral protein that is tightly associated with the membrane. Surface biotinylation experiments combined with western blot analyses and confocal immunolocalization revealed that SmGBP localized to the surface membranes of adult male schistosomes, especially the dorsal tubercles. In contrast, we detected little or no expression of SmGBP either in the females or larval stages. A comparative quantitative PCR analysis confirmed that the level of SmGBP expression is several-fold higher in male worms than cercariae, and it is barely detectable in adult females. Together, the results identify SmGBP as a new type of schistosome glutamate receptor that is both gender- and stage-specific. The high-level expression of this protein in the male tubercles suggests a possible role in host-parasite interaction.

Spotlight on the in vitro effect of artemisinin-naphthoquine phosphate on Schistosoma mansoni and its snail host Biomphalaria alexandrina

Malaria and schistosomiasis are the two most important parasitic diseases in the tropics and sub-tropics with geographic overlap. Efforts have been made for developing new schistosomicidal drugs, or testing existing drugs originally used for non-related diseases. The antimalarial artemisinin-naphthoquine phosphate combination (CO-ArNp) was recently reported to be a promising novel antischistosomal therapy with potent in vivo activity against Schistosoma mansoni. In this work, we report the in vitro dose- and time-response effect of CO-ArNp against the Egyptian strain of S. mansoni, and its snail host, Biomphalaria alexandrina. Incubation of adult S. mansoni with CO-ArNp at 40 or 20 μg/ml for 48 or 72 h killed all worms. Exposure of S. mansoni miracidia and cercariae to the molluscicidal LC50 of CO-ArNp (16.8 μg/ml) resulted in 100% mortality of the free larval stages within 90 and 15 min, respectively. Moreover, incubation of adult B. alexandrina snails with this drug combination killed all snails at 40 μg/ml within 24h. Scanning electron microscope revealed marked morphological and tegumental alterations on the different stages of the parasite and its snail soft tissue. Our study highlights the schistosomicidal and molluscicidal effects of artemisinin-naphthoquine phosphate. No doubt more studies are needed to clarify its potential value to control schistosomiasis.

In vivo effect of single oral dose of artemether against early juvenile stages of Schistosoma mansoni Egyptian strain.

The current treatment and control of schistosomiasis, rely on a single drug, praziquantel, although, it has minor activity against juvenile stages of the parasite. Studies have shown that artemether (ART) exhibits effects against juveniles of Schistosoma mansoni Liberian and Puerto Rican strains, Schistosoma japonicum and Schistosoma haematobium. Aiming to assess the in vivo activity of single oral dose of ART against early juvenile stages of S. mansoni Egyptian strain, this study was established. Mice were treated with ART (400 mg/kg) at two time points evenly spaced over the period of larval development (7 and 21 days post-infection; pi), and a third treatment point (day 49 pi) was included to elucidate when susceptibility decreases. Administration of ART on day 7 pi reduced the total worm burden by 85.94%. The greatest reductions were seen when treatment was given on day 21 pi, with total and female worm burden reductions of 91.52% and 90.57%, respectively, and cessation of oviposition. Similar dose given on day 49 pi reduced total worm burden by 55.17% and female worm burden by 66.51%. Moreover, it induced significant reduction in the tissue egg load and significant alterations in the oogram pattern with decreased immature eggs and increased dead eggs. Antipathological activities were evident in significant reductions in granulomata count and diameter. In conclusion, ART exhibits major in vivo schistosomicidal effects against the early larval migratory stages of S. mansoni Egyptian strain, mainly the 21-day old schistosomula, hence preventing disease progression and morbidity.

The epidemiology of Giardia intestinalis assemblages A and B among Egyptian children with diarrhea: A PCR-RFLP-based approach

Background The protozoan parasite Giardia intestinalis is a common childhood infection in developing countries that causes diarrheal illness. The majority of G. intestinalis isolates from humans are grouped into two distinct genetic assemblages A and B. The molecular epidemiological studies on G. intestinalis assemblages in humans are limited in Egypt. Objective This study was conducted to estimate the detection rate of G. intestinalis infection among a cohort of children suffering from diarrhea in the Dakahalia governorate, Egypt, and to correlate between clinical giardiasis and Giardia spp. assemblages in positive stool samples by PCR-restriction fragment length polymorphism (PCR-RFLP). Participants and methods A total of 311 diarrheal stool samples were examined microscopically for Giardia spp. infection. DNA samples were isolated from the stools of 103 (33.12%) positive samples with G. intestinalis, amplified with PCR, and digested with the XhoI enzyme for RFLP. Results Of the 103 samples, 64 (62.14%) were found to be assemblage B, whereas 32 samples (31.07%) belonged to assemblage A. Mixed genotype A and B was present in three samples (2.91%), and four samples (3.88%) were of undetermined Giardia spp. assemblage. The detection rate of assemblage B was higher in samples from children with persistent diarrhea, whereas assemblage A detection rate was higher in samples from acute diarrhea. Conclusion G. intestinalis causing diarrhea in children in the Dakahalia governorate, Egypt, predominantly belongs to assemblage B, indicating that human-to-human method of infection is more common than zoonotic method.

Schistosoma mansoni infection in a fishermen community, the Lake Manzala region-Egypt

Abstract Objective To determine the prevalence of schistosomiasis in the fishermen community in Egypt. Methods A cross-sectional survey for schistosomiasis mansoni was conducted among 150 fishermen and their families from January to November 2013. Faecal samples were examined by Kato Katz method and formalin-ether concentration technique. Malacological survey was conducted to identify infection of the snail intermediate host by larval stage of Schistosoma mansoni . Snails were collected and checked for shedding of cercariae after light exposure. Results Overall prevalence of infection was 26.6% with an intensity of (42.7依7.2) ova/g of stool. Infection was common in male and significantly increased in the age of 20-40 years. Praziquantel-treated individuals had a high significant decrease in intensity (27.2依2.4) ova/g of stool than those with no treatment history. Biomphalaria alexandrina snail was infected with Schistosoma mansoni particularly in warm seasons and mice infection was established successfully from the shed cercariae, moreover adult worms were obtained via portal perfusion of the infected mice. Conclusions Findings indicated the endemicity of schistosomiasis mansoni in Lake Manzala region, therefore, appropriate integrated control measures are needed among fishermen including health education, environmental sanitation, periodic screening and mass treatment with praziquantel.