Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis.
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ABSTRACT:
SUBMITTER: Gauci C
PROVIDER: S-EPMC128094 | biostudies-literature | 2002 Jul
REPOSITORIES: biostudies-literature
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Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis.
Gauci Charles C Merli Michael M Muller Volker V Chow Conan C Yagi Kinpei K Mackenstedt Ute U Lightowlers Marshall W MW
Infection and immunity 20020701 7
Alveolar and cystic hydatidosis are caused by infection with the larval stages of Echinococcus multilocularis and Echinococcus granulosus, respectively. A host-protective antigen has been identified in E. granulosus. Here we identify the presence of a closely related protein in E. multilocularis, characterize and express a cDNA encoding the antigen (designated EM95), determine the structure of the em95 gene, and demonstrate that the EM95 recombinant protein can be used to induce significant leve ...[more]
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Project description:BackgroundSerotonin is a phylogenetically ancient molecule that is widely distributed in most metazoans, including flatworms. In addition to its role as a neurotransmitter, serotonin acts as a morphogen and regulates developmental processes. Although several studies have focused on the serotonergic nervous system in parasitic flatworms, little is known on the role of serotonin in flatworm development.MethodsTo study the effects of serotonin on proliferation and development of the cestode Echinococcus multilocularis, we cloned the genes encoding the E. multilocularis serotonin transporter (SERT) and tryptophan hydroxylase (TPH), analyzed gene expression by transcriptome analysis and whole mount in situ hybridization (WMISH) and performed cell culture experiments.ResultsWe first characterized orthologues encoding the SERT and TPH, the rate-limiting enzyme in serotonin biosynthesis. WMISH and transcriptomic analyses indicated that the genes for both SERT and TPH are expressed in the parasite nervous system. Long-term treatment of parasite stem cell cultures with serotonin stimulated development towards the parasite metacestode stage. Mature metacestode vesicles treated with serotonin showed increased rates of incorporation of the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), indicating stimulated cell proliferation. In contrast, treatment with the selective serotonin reuptake inhibitor paroxetine strongly affected the viability of parasite cells. Paroxetine also caused structural damage in metacestode vesicles, suggesting that serotonin transport is crucial for the integrity of parasite vesicles.ConclusionsOur results indicate that serotonin plays an important role in E. multilocularis development and proliferation, providing evidence that the E. multilocularis SERT and TPH are expressed in the nervous system of the protoscolex. Our results further suggest that the E. multilocularis SERT has a secondary role outside the nervous system that is essential for parasite integrity and survival. Since serotonin stimulated E. multilocularis metacestode development and proliferation, serotonin might also contribute to the formation and growth of the parasite in the liver.
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Project description:Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH(2)-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.
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Project description:Alveolar echinococcosis (AE) is a severe disease caused by the larval stage of the tapeworm Echinococcus multilocularis Current chemotherapeutic treatment options based on benzimidazoles are of limited effectiveness, which underlines the need to find new antiechinococcosis drugs. Metformin is an antihyperglycemic and antiproliferative agent that shows activity against the related parasite Echinococcus granulosus Hence, we assessed the in vitro and in vivo effects of the drug on E. multilocularis Metformin exerted significant dose-dependent killing effects on in vitro cultured parasite stem cells and protoscoleces and significantly reduced the dedifferentiation of protoscoleces into metacestodes. Likewise, oral administration of metformin (50?mg/kg of body weight/day for 8?weeks) was effective in achieving a significant reduction of parasite weight in a secondary murine AE model. Our results revealed mitochondrial membrane depolarization, activation of Em-AMPK, suppression of Em-TOR, and overexpression of Em-Atg8 in the germinal layer of metformin-treated metacestode vesicles. The opposite effects on the level of active Em-TOR in response to exogenous insulin and rapamycin suggest that Em-TOR is part of the parasite's insulin signaling pathway. Finally, the presence of the key lysosomal pathway components, through which metformin reportedly acts, was confirmed in the parasite by in silico assays. Taken together, these results introduce metformin as a promising candidate for AE treatment. Although our study highlights the importance of those direct mechanisms by which metformin reduces parasite viability, it does not necessarily preclude any additional systemic effects of the drug that might reduce parasite growth in vivo.
| S-EPMC7449199 | biostudies-literature
Project description:BACKGROUND: The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system. RESULTS: We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-? and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm. CONCLUSIONS: We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages.
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Project description:Natural infection with larval Echinococcus multilocularis was recognized in one of eight Norway rats, Rattus norvegicus, caught indoors in 2009 in Ebetsu, Hokkaido, northern Japan. Cystic lesions were found in the right median and lateral lobes of the liver, with numerous alveolar cysts in the periphery of the lesions. Protoscolices were formed within large cysts. The laminated layers of the cysts were positive for PAS staining. Nested PCR using the primers specific for Taenia mitochondrial 12S rDNA yielded a 250-bp product, and the sequence of the PCR product matched that of E. multilocularis isolates from Hokkaido and Germany. This is the third natural alveolar hydatidosis in R. norvegicus in Japan.
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Project description:BACKGROUND: The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS: Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS: Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.
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Project description:BackgroundAlveolar echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus multilocularis. Many species of small mammals, including arvicolid rodents or Ochotona spp., are natural intermediate hosts of the cestode. The main aim of this study was to identify natural intermediate hosts of E. multilocularis in Chenaran County, Razavi Khorasan Province, northeastern Iran, where the prevalence of infected wild and domestic carnivores is high.Methodology/principal findingsA program of trapping was carried out in five villages in which this cestode was reported in carnivores. The livers of 85 small mammals were investigated for the presence of E. multilocularis infection using multiplex PCR of mitochondrial genes. Infections were identified in 30 specimens: 23 Microtus transcaspicus, three Ochotona rufescens, two Mus musculus, one Crocidura gmelini, and one Apodemus witherbyi.Conclusions/significanceA range of small mammals therefore act as natural intermediate hosts for the transmission of E. multilocularis in Chenaran County, and the prevalence suggested that E. multilocularis infection is endemic in this region. The existence of the life cycle of this potentially lethal cestode in the vicinity of human habitats provides a significant risk of human infection.
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Project description:This study aimed to find out the occurrence of Echinococcus multilocularis in foxes in Erzurum province, the highest endemic region for human alveolar echinococcosis in Turkey. The sedimentation and counting technique was used to reveal adult Echinococcus spp. in the intestines of foxes. One out of the 10 foxes was infected with E. multilocularis. The adult worms were analyzed morphologically and molecularly and were confirmed to be E. multilocularis by species-specific PCR. Pairwise comparisons between the 12S rRNA sequences of the E. multilocularis isolate from Erzurum and other E. multilocularis isolates showed 100% similarity of the Erzurum isolate with European isolates. With this study, the presence of E. multilocularis in a fox in Erzurum was confirmed by PCR, and molecular identification of E. multilocularis is reported for the first time in Turkey.
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