RESUMO
Subarachnoid neurocysticercosis (SANCC) is caused by an abnormally transformed form of the metacestode or larval form of the tapeworm Taenia solium. In contrast to vesicular parenchymal and ventricular located cysts that contain a viable scolex and are anlage of the adult tapeworm, the subarachnoid cyst proliferates to form aberrant membranous cystic masses within the subarachnoid spaces that cause mass effects and acute and chronic arachnoiditis. How subarachnoid cyst proliferates and interacts with the human host is poorly understood, but parasite stem cells (germinative cells) likely participate. RNA-seq analysis of the subarachnoid cyst bladder wall compared to the bladder wall and scolex of the vesicular cyst revealed that the subarachnoid form exhibits activation of signaling pathways that promote proliferation and increased lipid metabolism. These adaptions allow growth in a nutrient-limited cerebral spinal fluid. In addition, we identified therapeutic drug targets that would inhibit growth of the parasite, potentially increase effectiveness of treatment, and shorten its duration.
Assuntos
Neurocisticercose , Espaço Subaracnóideo , Taenia solium , Animais , Taenia solium/genética , Neurocisticercose/parasitologia , Neurocisticercose/genética , Espaço Subaracnóideo/metabolismo , Humanos , Perfilação da Expressão Gênica , Transcriptoma , Proliferação de Células , Cistos/genética , Cistos/parasitologia , Cistos/metabolismoRESUMO
Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.
Assuntos
Glutationa Transferase , Taenia solium , Taenia solium/genética , Taenia solium/enzimologia , Animais , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Glutationa Transferase/química , Humanos , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Regiões Promotoras Genéticas/genéticaRESUMO
Human cysticercosis by Taenia solium is the major cause of neurological illness in countries of Africa, Southeast Asia, and the Americas. Publication of four cestode genomes (T. solium, Echinococcus multilocularis, E. granulosus and Hymenolepis microstoma) in the last decade, marked the advent of novel approaches on the study of the host-parasite molecular crosstalk for cestode parasites of importance for human and animal health. Taenia crassiceps is another cestode parasite, closely related to T. solium, which has been used in numerous studies as an animal model for human cysticercosis. Therefore, characterization of the T. crassiceps genome will also contribute to the understanding of the human infection. Here, we report the genome of T. crassiceps WFU strain, reconstructed to a noncontiguous finished resolution and performed a genomic and differential expression comparison analysis against ORF strain. Both strain genomes were sequenced using Oxford Nanopore (MinION) and Illumina technologies, achieving high quality assemblies of about 107 Mb for both strains. Dotplot comparison between WFU and ORF demonstrated that both genomes were extremely similar. Additionally, karyotyping results for both strains failed to demonstrate a difference in chromosome composition. Therefore, our results strongly support the concept that the absence of scolex in the ORF strain of T. crassiceps was not the result of a chromosomal loss as proposed elsewhere. Instead, it appears to be the result of subtle and extensive differences in the regulation of gene expression. Analysis of variants between the two strains identified 2,487 sites with changes distributed in 31 of 65 scaffolds. The differential expression analysis revealed that genes related to development and morphogenesis in the ORF strain might be involved in the lack of scolex formation.
Assuntos
Cisticercose , Taenia solium , África , Animais , Cisticercose/veterinária , Modelos Animais de Doenças , Genômica , Humanos , Taenia solium/genéticaRESUMO
Neurocysticercosis caused by Taenia solium larvae is a neglected disease that persists in several countries, including Mexico, and causes a high disability-adjusted life year burden. Neuroimaging tools such as computed tomography and magnetic resonance imaging are the most efficient for its detection, but low availability and high costs in most endemic regions limit their use. Serological methods such as lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot antibody detection and monoclonal antibody-based enzyme-linked immunosorbent assays for HP10 antigen detection have been useful in supporting the diagnosis of this disease. We evaluated three T. solium recombinant antigens: glutathione transferase of 26 kDa (Ts26GST); thioredoxin-1 (TsTrx-1), and endophilin B1 (TsMEndoB1) by EITB. These are antigenic proteins antigenic, abundant in excretion/secretion products of the parasite, and do not cross-react with homologous host proteins. Ts26GST and TsTrx-1 showed sensitivity of 79 and 88%, specificity of 86 and 97%, PPV of 83 and 97% and NPV of 82 and 91%, respectively, for neurocysticercosis diagnosis. The recombinant antigens allowed the diagnosis of 70% (Ts26GST) and 80% (TsTrx-1) of patients having only one cysticercus. Further studies on specific regions of these proteins could improve T. solium diagnostics.
Assuntos
Neurocisticercose , Taenia solium , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/genética , Ensaio de Imunoadsorção Enzimática/métodos , Glutationa Transferase/genética , Humanos , Neurocisticercose/diagnóstico , Neurocisticercose/parasitologia , Sensibilidade e Especificidade , Taenia solium/genética , Tiorredoxinas/genéticaRESUMO
Neurocysticercosis (NCC), the infection of the nervous system by the cystic larvae of Taenia solium, is a highly pleomorphic disease because of differences in the number and anatomical location of lesions, the viability of parasites, and the severity of the host immune response. Most patients with parenchymal brain NCC present with few lesions and a relatively benign clinical course, but massive forms of parenchymal NCC can carry a poor prognosis if not well recognized and inappropriately managed. We present the main presentations of massive parenchymal NCC and their differential characteristics.
Assuntos
Encéfalo/parasitologia , Neurocisticercose/diagnóstico , Taenia solium/fisiologia , Animais , Encéfalo/diagnóstico por imagem , Diagnóstico Diferencial , Humanos , Neurocisticercose/diagnóstico por imagem , Neurocisticercose/parasitologia , Neurocisticercose/terapia , Taenia solium/genética , Taenia solium/isolamento & purificaçãoRESUMO
BACKGROUND: The pork tapeworm (Taenia solium) is a parasitic helminth that imposes a major health and economic burden on poor rural populations around the world. As recognized by the World Health Organization, a key barrier for achieving control of T. solium is the lack of an accurate and validated simulation model with which to study transmission and evaluate available control and elimination strategies. CystiAgent is a spatially-explicit agent based model for T. solium that is unique among T. solium models in its ability to represent key spatial and environmental features of transmission and simulate spatially targeted interventions, such as ring strategy. METHODS/PRINCIPAL FINDINGS: We validated CystiAgent against results from the Ring Strategy Trial (RST)-a large cluster-randomized trial conducted in northern Peru that evaluated six unique interventions for T. solium control in 23 villages. For the validation, each intervention strategy was replicated in CystiAgent, and the simulated prevalences of human taeniasis, porcine cysticercosis, and porcine seroincidence were compared against prevalence estimates from the trial. Results showed that CystiAgent produced declines in transmission in response to each of the six intervention strategies, but overestimated the effect of interventions in the majority of villages; simulated prevalences for human taenasis and porcine cysticercosis at the end of the trial were a median of 0.53 and 5.0 percentages points less than prevalence observed at the end of the trial, respectively. CONCLUSIONS/SIGNIFICANCE: The validation of CystiAgent represented an important step towards developing an accurate and reliable T. solium transmission model that can be deployed to fill critical gaps in our understanding of T. solium transmission and control. To improve model accuracy, future versions would benefit from improved data on pig immunity and resistance, field effectiveness of anti-helminthic treatment, and factors driving spatial clustering of T. solium infections including dispersion and contact with T. solium eggs in the environment.
Assuntos
Cisticercose/transmissão , Cisticercose/veterinária , Doenças dos Suínos/transmissão , Taenia solium/fisiologia , Zoonoses/transmissão , Animais , Cisticercose/epidemiologia , Cisticercose/parasitologia , Modelos Epidemiológicos , Feminino , Humanos , Peru/epidemiologia , Estudos Prospectivos , População Rural/estatística & dados numéricos , Análise Espacial , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/parasitologia , Taenia solium/genética , Taenia solium/isolamento & purificação , Zoonoses/epidemiologia , Zoonoses/parasitologiaRESUMO
Cysticerci of Taenia solium cause cysticercosis, with neurocysticercosis (NCC) as the major pathology. Sensible and specific recombinant antigens would be an source of antigen for immunodiagnosis. The objective of this work was the molecular characterization and evaluation, of three news recombinant antigens (TsF78, TsP43 and TsC28), obtained by screening of a Taenia solium cDNA library. The three cDNA were analysed by bioinformatic programs, subcloned and expresed. The purified proteins were evaluated in ELISA using cyst fluid as control. TsF78 is filamina, TsP43 a peroxidase and TsC28 collagen XV. The sensitivity and specificity of the recombinant proteins were; TsF78 93.8 % and 95.0 %, TsP62 91.7 % and 93.3 %, TsC28 85.4 % and 93.3 %, respectively, while the cyst fluid showed a sensitivity of 87.5 % and a specificity of 76.7 %. Given its high sensitivity and specificity, the recombinant proteins TsF78 and TsP62 could be used in the diagnosis of cysticercosis.
Assuntos
Antígenos de Helmintos/imunologia , Cisticercose/diagnóstico , Testes Imunológicos , Proteínas Recombinantes/imunologia , Taenia solium/imunologia , Teníase/diagnóstico , Animais , Antígenos de Helmintos/genética , Estudos de Casos e Controles , Cisticercose/imunologia , Cisticercose/microbiologia , Humanos , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Taenia solium/genética , Teníase/imunologia , Teníase/microbiologiaRESUMO
BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Calreticulina/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/patologia , Calreticulina/genética , Calreticulina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Células HeLa , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Taenia solium/genéticaRESUMO
We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.
Assuntos
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Mitocôndrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cysticercus/genética , Cysticercus/isolamento & purificação , Cysticercus/metabolismo , Citosol/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Fases de Leitura Aberta , Coelhos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMO
Several studies have been performed to determine specific antigens for the diagnosis of tapeworms. One of these antigens is Tso31, which is used to differentiate Taenia solium and Taenia saginata in human feces. The aim of the present work was the molecular characterization of this protein in different tapeworm specimens collected in Peru: T. omisa (n = 6), T. hydatigena (n = 7), T. taeniaeformis (n = 4), T. pisiformes (n = 1), T. multiceps (n = 7), and T. solium (n = 10). Total DNA was extracted from each proglottid using a commercial DNA kit for tissue. A nested PCR was used to amplify a fragment of the previously described oncosphere-specific protein Tso31 gene. The nested PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized after ethidium bromide staining. All nested PCR-positive products were sequenced and their sequences were compared. Of all the tapeworms analyzed, only T. solium and T. multiceps amplified the Tso31 gene. All sequences were identical for each species. Our T. solium Tso31 showed 100% similarity when compared with published GenBank sequences. The difference between T. solium and T. multiceps Tso31 samples was 8.1%. In conclusion, our results show that the tsol31 gene is not exclusive to T. solium.
Assuntos
Antígenos de Helmintos/genética , Taenia saginata/genética , Taenia solium/genética , Teníase/diagnóstico , Animais , Sequência de Bases , DNA , DNA de Helmintos/genética , Fezes/parasitologia , Humanos , Peru , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Taenia , Taenia saginata/metabolismo , Taenia solium/metabolismo , Teníase/parasitologiaRESUMO
Neurocysticercosis (NCC), caused by Taenia solium larvae that reside in the central nervous system, results in serious public health and medical issues in many regions of the world. Current diagnosis of NCC is complex requiring both serology and costly neuroimaging of parasitic cysts in the brain. This diagnostic pipeline can be problematic in resource-constrained settings. There is an unmet need for a highly sensitive and clinically informative diagnostic test to complement the present diagnostic approaches. Here, we report that T. solium-derived cell-free DNA is readily detectable in the urine of patients with the subarachnoid and parenchymal forms of NCC, and discuss the potential utility of this approach in enhancing and refining T. solium diagnostics.
Assuntos
Ácidos Nucleicos Livres/genética , Disfunção Cognitiva/parasitologia , DNA de Helmintos/genética , Neurocisticercose/parasitologia , Taenia solium/genética , Animais , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/urina , Sistema Nervoso Central/parasitologia , Sistema Nervoso Central/fisiopatologia , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/fisiopatologia , DNA de Helmintos/sangue , DNA de Helmintos/urina , Humanos , Larva/genética , Neurocisticercose/diagnóstico por imagem , Neurocisticercose/fisiopatologia , Neuroimagem/métodos , Peru , Reação em Cadeia da Polimerase/métodos , Taenia solium/isolamento & purificaçãoRESUMO
BACKGROUND: Due to the relative short life span and the limited spatial movement, porcine cysticercosis is an excellent indicator of current local active transmission. The aim of this study was to map at province-level, the occurrence of T. solium and T. asiatica in pigs and areas at risk of transmission to pigs in East and Southeast Asia, based on the density of extensive pig production systems and confirmed reports of porcine cysticercosis. METHODS: This study covered East and Southeast Asia, which consist of the following countries: Brunei, Cambodia, China, East Timor, Indonesia, Japan, Laos, Malaysia, Mongolia, Myanmar, North Korea, Philippines, Singapore, South Korea, Thailand and Vietnam. Literature searches were carried out to identify current epidemiological data on the occurrence of porcine cysticercosis caused by T. solium and T. asiatica infections. Modelled densities of pigs in extensive production systems were mapped and compared to available data on porcine cysticercosis. RESULTS: Porcine cysticercosis was confirmed to be present during the period 2000 to 2018 in eight out of the 16 countries included in this study. Taenia solium porcine cysticercosis was confirmed from all eight countries, whereas only one country (Laos) could confirm the presence of T. asiatica porcine cysticercosis. Province-level occurrence was identified in five countries (Cambodia, Indonesia, Laos, Myanmar, and Vietnam) across 19 provinces. Smallholder pig keeping is believed to be widely distributed throughout the region, with greater densities predicted to occur in areas of China, Myanmar, Philippines and Vietnam. CONCLUSIONS: The discrepancies between countries reporting taeniosis and the occurrence of porcine cysticercosis, both for T. solium and T. asiatica, suggests that both parasites are underreported. More epidemiological surveys are needed to determine the societal burden of both parasites. This study highlights a straightforward approach to determine areas at risk of porcine cysticercosis in the absence of prevalence data.
Assuntos
Cisticercose/parasitologia , Cisticercose/veterinária , Doenças dos Suínos/parasitologia , Taenia solium/isolamento & purificação , Taenia/isolamento & purificação , Animais , Sudeste Asiático/epidemiologia , Cisticercose/epidemiologia , Ásia Oriental/epidemiologia , Humanos , Suínos , Doenças dos Suínos/epidemiologia , Taenia/classificação , Taenia/genética , Taenia solium/classificação , Taenia solium/genéticaRESUMO
The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.
Assuntos
Extratos Celulares/análise , Cysticercus/metabolismo , Proteoma/análise , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , Taenia solium/metabolismo , Animais , Antígenos de Helmintos , Sistema Nervoso Central/parasitologia , Cromatografia Líquida , Cysticercus/genética , Cysticercus/imunologia , Países em Desenvolvimento , Perfilação da Expressão Gênica , Humanos , Larva/metabolismo , Músculo Esquelético/parasitologia , Doenças Negligenciadas/parasitologia , Neurocisticercose/diagnóstico , Neurocisticercose/parasitologia , Proteômica , Saúde Pública , Taenia solium/genética , Taenia solium/imunologia , Teníase/diagnóstico , Teníase/parasitologia , Zoonoses/parasitologiaRESUMO
The aim of this study was to demonstrate the presence of Taenia solium eggs in beetles collected from sources within the natural environment through molecular techniques. Fifty-four pools of beetles were collected in three villages in Piura, Peru. DNA was extracted using the FastDNA spin kit for soil. Molecular identification of Taenia species was then performed through partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Finally, positive samples were sequenced to determine the tapeworm species. Seven positive samples were obtained through polymerase chain reaction amplification. Sequencing confirmed that two samples were from T. solium and three samples were from Taenia hydatigena. The other two samples could not be specifically identified. Our findings demonstrate that dung beetles ingest T. solium and T. hydatigena eggs under natural conditions and suggest that beetles may play a role in the dynamics of transmission of these cestodes.
Assuntos
Besouros/parasitologia , DNA de Helmintos/genética , Óvulo , Taenia solium/isolamento & purificação , Teníase/transmissão , Animais , DNA de Helmintos/isolamento & purificação , Contagem de Ovos de Parasitas , Peru/epidemiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Taenia/genética , Taenia/isolamento & purificação , Taenia solium/genética , Teníase/epidemiologia , Teníase/parasitologiaRESUMO
Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were â¼48 and â¼50â¯kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091â¯mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.
Assuntos
Antígenos de Helmintos/imunologia , Cisticercose/veterinária , Fosfopiruvato Hidratase/imunologia , Doenças dos Suínos/diagnóstico , Taenia solium/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/biossíntese , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/metabolismo , Biologia Computacional , Intervalos de Confiança , Cisticercose/diagnóstico , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Vetores Genéticos , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Curva ROC , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Alinhamento de Sequência , Células Sf9 , Espectrofotometria/veterinária , Suínos , Doenças dos Suínos/parasitologia , Taenia solium/classificação , Taenia solium/genética , Taenia solium/imunologiaRESUMO
BACKGROUND: We have previously reported that progesterone (P4) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. METHODS: By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. RESULTS: Our results show that P4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P4-binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. CONCLUSION: Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.
Assuntos
Interações Hospedeiro-Parasita , Progesterona/metabolismo , Receptores de Progesterona/genética , Taenia solium/crescimento & desenvolvimento , Taenia solium/genética , Animais , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Simulação de Acoplamento Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Suínos , Taenia solium/efeitos dos fármacosRESUMO
The adult Taenia solium, the pork tapeworm, usually lives as a single worm in the small intestine of humans, its only known definitive host. Mechanisms of genetic variation in T. solium are poorly understood. Using three microsatellite markers previously reported [1], this study explored the genetic variability of T. solium from cysts recovered from experimentally infected pigs. It then explored the genetic epidemiology and transmission in naturally infected pigs and adult tapeworms recovered from human carriers from an endemic rural community in Peru. In an initial study on experimental infection, two groups of three piglets were each infected with proglottids from one of two genetically different tapeworms for each of the microsatellites. After 7 weeks, pigs were slaughtered and necropsy performed. Thirty-six (92.3%) out of 39 cysts originated from one tapeworm, and 27 (100%) out of 27 cysts from the other had exactly the same genotype as the parental tapeworm. This suggests that the microsatellite markers may be a useful tool for studying the transmission of T. solium. In the second study, we analyzed the genetic variation of T. solium in cysts recovered from eight naturally infected pigs, and from adult tapeworms recovered from four human carriers; they showed genetic variability. Four pigs had cysts with only one genotype, and four pigs had cysts with two different genotypes, suggesting that multiple infections of genetically distinct parental tapeworms are possible. Six pigs harbored cysts with a genotype corresponding to one of the identified tapeworms from the human carriers. In the dendrogram, cysts appeared to cluster within the corresponding pigs as well as with the geographical origin, but this association was not statistically significant. We conclude that genotyping of microsatellite size polymorphisms is a potentially important tool to trace the spread of infection and pinpoint sources of infection as pigs spread cysts with a shared parental genotype.
Assuntos
Cisticercose/veterinária , Repetições de Microssatélites/genética , Taenia solium/genética , Teníase/veterinária , Animais , Cisticercose/parasitologia , Cisticercose/transmissão , Cysticercus/genética , Cysticercus/isolamento & purificação , Cistos/parasitologia , Modelos Animais de Doenças , Feminino , Variação Genética/genética , Genótipo , Masculino , Peru , Sus scrofa , Suínos , Doenças dos Suínos/parasitologia , Taenia solium/isolamento & purificação , Teníase/parasitologia , Teníase/transmissãoRESUMO
Taeniids exhibit a great adaptive plasticity, which facilitates their establishment, growth, and reproduction in a hostile inflammatory microenvironment. Transforming Growth Factor-ß (TGFß), a highly pleiotropic cytokine, plays a critical role in vertebrate morphogenesis, cell differentiation, reproduction, and immune suppression. TGFß is secreted by host cells in sites lodging parasites. The role of TGFß in the outcome of T. solium and T. crassiceps cysticercosis is herein explored. Homologues of the TGFß family receptors (TsRI and TsRII) and several members of the TGFß downstream signal transduction pathway were found in T. solium genome, and the expression of Type-I and -II TGFß receptors was confirmed by RT-PCR. Antibodies against TGFß family receptors recognized cysticercal proteins of the expected molecular weight as determined by Western blot, and different structures in the parasite external tegument. In vitro, TGFß promoted the growth and reproduction of T. crassiceps cysticerci and the survival of T. solium cysticerci. High TGFß levels were found in cerebrospinal fluid from untreated neurocysticercotic patients who eventually failed to respond to the treatment (P = 0.03) pointing to the involvement of TGFß in parasite survival. These results indicate the relevance of TGFß in the infection outcome by promoting cysticercus growth and treatment resistance.
Assuntos
Cysticercus/imunologia , Interações Hospedeiro-Parasita/imunologia , Neurocisticercose/imunologia , Taenia solium/imunologia , Fator de Crescimento Transformador beta/imunologia , Receptores de Ativinas/genética , Receptores de Ativinas/imunologia , Receptores de Ativinas/metabolismo , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/metabolismo , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Cysticercus/genética , Cysticercus/metabolismo , Modelos Animais de Doenças , Resistência a Medicamentos/imunologia , Genoma Helmíntico/imunologia , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neurocisticercose/líquido cefalorraquidiano , Neurocisticercose/tratamento farmacológico , Neurocisticercose/parasitologia , Transdução de Sinais/imunologia , Suínos , Taenia solium/genética , Taenia solium/metabolismo , Fator de Crescimento Transformador beta/líquido cefalorraquidiano , Fator de Crescimento Transformador beta/metabolismoRESUMO
Tapeworms Taenia solium and Taenia saginata are the causative agents of taeniasis/cysticercosis. These are diseases with high medical and veterinary importance due to their impact on public health and rural economy in tropical countries. The re-emergence of T. solium as a result of human migration, the economic burden affecting livestock industry, and the large variability of symptoms in several human cysticercosis, encourage studies on genetic diversity, and the identification of these parasites with molecular phylogenetic tools. Samples collected from the Ecuadorian provinces: Loja, Guayas, Manabí, Tungurahua (South), and Imbabura, Pichincha (North) from 2000 to 2012 were performed under Maximum Parsimony analyses and haplotype networks using partial sequences of mitochondrial DNA, cytochrome oxidase subunit I (COI) and NADH subunit I (NDI), from Genbank and own sequences of Taenia solium and Taenia saginata from Ecuador. Both species have shown reciprocal monophyly, which confirms its molecular taxonomic identity. The COI and NDI genes results suggest phylogenetic structure for both parasite species from south and north of Ecuador. In T. solium, both genes gene revealed greater geographic structure, whereas in T. saginata, the variability for both genes was low. In conclusion, COI haplotype networks of T. solium suggest two geographical events in the introduction of this species in Ecuador (African and Asian lineages) and occurring sympatric, probably through the most common routes of maritime trade between the XV-XIX centuries. Moreover, the evidence of two NDI geographical lineages in T. solium from the north (province of Imbabura) and the south (province of Loja) of Ecuador derivate from a common Indian ancestor open new approaches for studies on genetic populations and eco-epidemiology.
Assuntos
DNA Mitocondrial , Variação Genética , Taenia saginata/genética , Taenia solium/genética , Teníase/parasitologia , Animais , Equador/epidemiologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Fluxo Gênico , Haplótipos , NADH Desidrogenase/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Taenia saginata/classificação , Taenia solium/classificação , Teníase/epidemiologiaRESUMO
BACKGROUND: The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages. METHODOLOGY/PRINCIPAL FINDINGS: T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development--days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15-30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages. CONCLUSIONS/SIGNIFICANCE: This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite's immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.