RESUMO
Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.
Assuntos
Cestoides/metabolismo , Proteínas de Helminto/metabolismo , Sistema Nervoso/metabolismo , Receptores 5-HT1 de Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Echinococcus/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Hymenolepis/metabolismo , Receptores 5-HT1 de Serotonina/química , Receptores 5-HT1 de Serotonina/genética , Alinhamento de Sequência , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
BACKGROUND: Cestoda is a class of endoparasitic worms in the flatworm phylum (Platyhelminthes). During the course of their evolution cestodes have evolved some interesting aspects, such as their increased reproductive capacity. In this sense, they have serial repetition of their reproductive organs in the adult stage, which is often associated with external segmentation in a developmental process called strobilation. However, the molecular basis of strobilation is poorly understood. To assess this issue, an evolutionary comparative study among strobilated and non-strobilated flatworm species was conducted to identify genes and proteins related to the strobilation process. RESULTS: We compared the genomic content of 10 parasitic platyhelminth species; five from cestode species, representing strobilated parasitic platyhelminths, and five from trematode species, representing non-strobilated parasitic platyhelminths. This dataset was used to identify 1813 genes with orthologues that are present in all cestode (strobilated) species, but absent from at least one trematode (non-strobilated) species. Development-related genes, along with genes of unknown function (UF), were then selected based on their transcriptional profiles, resulting in a total of 34 genes that were differentially expressed between the larval (pre-strobilation) and adult (strobilated) stages in at least one cestode species. These 34 genes were then assumed to be strobilation related; they included 12 encoding proteins of known function, with 6 related to the Wnt, TGF-ß/BMP, or G-protein coupled receptor signaling pathways; and 22 encoding UF proteins. In order to assign function to at least some of the UF genes/proteins, a global gene co-expression analysis was performed for the cestode species Echinococcus multilocularis. This resulted in eight UF genes/proteins being predicted as related to developmental, reproductive, vesicle transport, or signaling processes. CONCLUSIONS: Overall, the described in silico data provided evidence of the involvement of 34 genes/proteins and at least 3 developmental pathways in the cestode strobilation process. These results highlight on the molecular mechanisms and evolution of the cestode strobilation process, and point to several interesting proteins as potential developmental markers and/or targets for the development of novel antihelminthic drugs.
Assuntos
Cestoides/crescimento & desenvolvimento , Cestoides/genética , Animais , Cestoides/classificação , Cestoides/metabolismo , Evolução Molecular , Perfilação da Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , FilogeniaRESUMO
Here we evaluate the potential of heavy metal accumulation of Proteocephalus macrophallus parasitizing the Butterfly Peacock Bass (Cichla ocellaris). A total of 19 fish specimens were collected. From the hosts, samples of intestine, liver, muscle, and parasites were taken. Heavy metal concentrations (Al, As, Ba, Cd, Cr, Fe, Hg, Mg, Mn, Ni, Pb, Ti, and Zn) were obtained using Atomic Absorption Spectrometry. All analyzed elements was found in higher concentrations in the parasites comparing to its host tissues. The bioconcentration factors were higher in the intestine, varying between 5.91 (Ti) to 8.00 (Ba), followed by the muscle, 1.88 (Mg) to 6.39 (Zn), and liver, 1.67 (Al) to 2.02 (Ba). These results show that at the infection site heavy metal concentrations are reduced, since the elements are absorbed directly from the intestinal wall by the parasites. In general, P. macrophallus presents a reasonable capacity of metal accumulation comparing to its hosts.
Assuntos
Cestoides/metabolismo , Ciclídeos/parasitologia , Monitoramento Ambiental/métodos , Intestinos/química , Metais Pesados/análise , Poluentes Químicos da Água/análise , Animais , Brasil , Cestoides/isolamento & purificação , Alimentos Marinhos/análiseRESUMO
Sex hormone receptors play critical roles in development and reproduction. However, it is not known whether they exist in Raillietina tapeworms, and if they do, whether they have a similar function to that in vertebrates. We examined the immunohistochemical distributions of androgen receptors (ARs), estrogen receptors (ERs), and progesterone receptors (PRs) in the tissues of two tapeworm species: Raillietina echinobothrida and Raillietina tetragona. Immunopositive ARs were found in the entire reproductive system of R. echinobothrida, including the testes, ovaries, and oocysts, and weakly immunopositive ERs and PRs were found in the testes, ovaries, and oocysts. Immunopositive ARs were also found throughout the entire reproductive system of R. tetragona, including the testes, ovaries, and oocysts, and weakly immunopositive ERs were in the testes and oocysts; the PRs were distributed in an immunonegative manner. The results show that androgens and their receptors play critical roles in reproductive system development in the two tapeworms. The immunoreactivity and tissue localizations of the sex hormone receptors suggest that, in both species, they have similar functions as in vertebrates, and modulate reproduction.
Assuntos
Cestoides/metabolismo , Androgênios/metabolismo , Animais , Hormônios Esteroides Gonadais/metabolismo , Imuno-Histoquímica , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Parasitic flatworms cause serious infectious diseases that affect humans and livestock in vast regions of the world, yet there are few effective drugs to treat them. Thioredoxin glutathione reductase (TGR) is an essential enzyme for redox homeostasis in flatworm parasites and a promising pharmacological target. We purified to homogeneity and characterized the TGR from the tapeworm Mesocestoides vogae (syn. M. corti). This purification revealed absence of conventional TR and GR. The glutathione reductase activity of the purified TGR exhibits a hysteretic behavior typical of flatworm TGRs. Consistently, M. vogae genome analysis revealed the presence of a selenocysteine-containing TGR and absence of conventional TR and GR. M. vogae thioredoxin and glutathione reductase activities were inhibited by 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole N2-oxide (VL16E), an oxadiazole N-oxide previously identified as an inhibitor of fluke and tapeworm TGRs. Finally, we show that mice experimentally infected with M. vogae tetrathyridia and treated with either praziquantel, the reference drug for flatworm infections, or VL16E exhibited a 28% reduction of intraperitoneal larvae numbers compared to vehicle treated mice. Our results show that oxadiazole N-oxide is a promising chemotype in vivo and highlights the convenience of M. vogae as a model for rapid assessment of tapeworm infections in vivo.
Assuntos
Cestoides/efeitos dos fármacos , Infecções por Cestoides/parasitologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxidiazóis/farmacologia , Sequência de Aminoácidos , Animais , Cestoides/metabolismo , Mesocestoides , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , NADH NADPH Oxirredutases/química , Homologia de Sequência de AminoácidosRESUMO
Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 µ g/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 µ g/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis.
Assuntos
Anticestoides/farmacologia , Bacillus thuringiensis/química , Toxinas Bacterianas/farmacologia , Cestoides/metabolismo , Animais , Anticestoides/química , Toxinas Bacterianas/química , Infecções por Cestoides/metabolismo , Infecções por Cestoides/prevenção & controle , Cães , Relação Dose-Resposta a Droga , Humanos , Locomoção/efeitos dos fármacos , Óvulo/metabolismo , Zoonoses/parasitologiaRESUMO
In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms.
Assuntos
Cestoides/metabolismo , Glutationa/metabolismo , Proteínas de Helminto/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Tiorredoxinas/metabolismo , Animais , Hidrazinas/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Oxirredução/efeitos dos fármacos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Anthelmintic molecules must reach their receptors inside target parasites to exert the pharmacological effect. Available data suggest that the main route of entry of antiparasitic drugs into helminth parasites would be through their external surface. However, it is unclear if trans-tegumental/cuticular penetration is the most important way of entry of benzimidazole (BZD) anthelmintics into their target parasites compared to oral ingestion. The relative involvement of active and passive transport mechanisms has not been defined. The goal of the work reported here was to determine the main processes involved in the entry of BZD anthelmintic molecules into the three main classes of helminth parasites. Adult specimens of Moniezia benedeni (cestode), Fasciola hepatica (trematode) and Ascaris suum (nematode) were incubated in Kreb's Ringer Tris buffer (pH 7.4, 37 degrees C) (1g parasite/10 ml incubation medium) for 15, 45, and 90 min, respectively, in the presence of a concentration gradient of either fenbendazole (FBZ), oxfendazole or triclabendazole sulphoxide (TCBZSO) (1-30 mol/ml, n=4). Dead helminth specimens were also incubated with the same drug concentration gradient. Specimens of F. hepatica with the oral route closed off by ligation were incubated with TCBZSO in the presence or absence of bovine serum albumin. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography analysis to measure drug/metabolite concentrations. Equivalent drug concentrations were measured within ligated and non-ligated liver flukes, demonstrating that BZD do mainly penetrate by trans-tegumental diffusion. The higher the concentration of BZD molecules in the incubation medium, the greater their concentration recovered within the helminth parasites. High correlation coefficients (>0.98) were obtained between initial drug concentration in the incubation medium and those measured inside the nematode, cestode, and trematode parasites. FBZ concentrations recovered from tissues of dead cestodes/nematodes over time were significantly greater compared to those measured in living parasites. These differences in drug diffusion may be related to the morphological/functional properties of the parasite's external surfaces. The outcome of the work reported here indicates that passive drug transfer through the external helminth surface is the main transport mechanism accounting for BZD accumulation into target parasites.
Assuntos
Anti-Helmínticos/metabolismo , Ascaris suum/metabolismo , Benzimidazóis/metabolismo , Cestoides/metabolismo , Fasciola hepatica/metabolismo , Animais , Transporte Biológico , Bovinos , Difusão , Feminino , Fenbendazol/metabolismo , Modelos Lineares , Moniezíase/parasitologia , Ovinos , Sulfóxidos/metabolismo , Suínos , TriclabendazolRESUMO
Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.
Assuntos
Ácidos Graxos/metabolismo , Proteínas de Helminto/química , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cestoides/química , Cestoides/metabolismo , Cromatografia de Afinidade , Líquido Cístico/metabolismo , Escherichia coli/genética , Ácidos Graxos/química , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Cinética , Ligantes , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de FluorescênciaRESUMO
The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol-water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5-210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb's Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol-water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation (r=0.87) between drug lipophilicity, expressed as octanol-water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument.