RESUMO
Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection.
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
Fatty acid-binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids de novo, FABPs have been proposed as essential proteins, and thus, as possible drug targets and/or carriers against these parasites. We performed data mining in Echinococcus multilocularis and Echinococcus granulosus genomes in order to test whether this family of proteins is more complex than previously reported. By exploring the genomes of E. multilocularis and E. granulosus, six genes coding for FABPs were found in each organism. In the case of E. granulosus, all of them have different coding sequences, whereas in E. multilocularis, two of the genes code for the same protein. Remarkably, one of the genes (in both cestodes) encodes a FABP with a C-terminal extension unusual for this family of proteins. The newly described genes present variations in their structure in comparison with previously described FABP genes in Echinococcus spp. The coding sequences for E. multilocularis were validated by cloning and sequencing. Moreover, differential expression patterns of FABPs were observed at different stages of the life cycle of E. multilocularis by exploring transcriptomic data from several sources. In summary, FABP family in cestodes is far more complex than previously thought and includes new members that seem to be only present in flatworms.
Assuntos
Echinococcus granulosus/genética , Echinococcus multilocularis/genética , Proteínas de Ligação a Ácido Graxo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Ácidos Graxos/metabolismo , Genoma de Protozoário/genética , Análise de Sequência , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
Cestode parasites cause neglected diseases, such as echinococcosis and cysticercosis, which represent a significant problem in human and animal health. Benzimidazoles and praziquantel are the only available drugs for chemotherapy and it is therefore important to identify new alternative drugs against cestode parasites. Histone deacetylases (HDACs) are validated drug targets for the treatment of cancer and other diseases, including neglected diseases. However, knowledge of HDACs in cestodes is very scarce. In this work, we investigated cestode HDACs as potential drug targets to develop new therapies against neglected diseases caused by cestodes. Here we showed the full repertoire of HDAC coding genes in several members of the class Cestoda. Between 6 and 7 zinc-dependent HDAC coding genes were identified in the genomes of species from Echinococcus, Taenia, Mesocestoides and Hymenolepis genera. We classified them as Class I and II HDACs and analyzed their transcriptional expression levels throughout developmental stages of Echinococcus spp. We confirmed for the first time the complete HDAC8 nucleotide sequences from Echinococcus canadensis G7 and Mesocestoides corti. Homology models for these proteins showed particular structural features which differentiate them from HDAC8 from Homo sapiens. Furthermore, we showed that Trichostatin A (TSA), a pan-HDAC inhibitor, decreases the viability of M. corti, alters its tegument and morphology and produces an increment of the total amount of acetylated proteins, including acetylated histone H4. These results suggest that HDAC from cestodes are functional and might play important roles on survival and development. The particular structural features observed in cestode HDAC8 proteins suggest that these enzymes could be selectively targeted. This report provides the basis for further studies on cestode HDAC enzymes and for discovery of new HDAC inhibitors for the treatment of neglected diseases caused by cestode parasites.
Assuntos
Cestoides/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/parasitologia , Animais , Cestoides/enzimologia , Infecções por Cestoides/tratamento farmacológico , Feminino , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos WistarRESUMO
BACKGROUND: Cestodes are a diverse group of parasites, some of them being agents of neglected diseases. In cestodes, little is known about the functional properties of G protein coupled receptors (GPCRs) which have proved to be highly druggable targets in other organisms. Notably, serotoninergic G-protein coupled receptors (5-HT GPCRs) play major roles in key functions like movement, development and reproduction in parasites. METHODOLOGY/PRINCIPAL FINDINGS: Three 5-HT GPCRs from Echinococcus granulosus and Mesocestoides corti were cloned, sequenced, bioinformatically analyzed and functionally characterized. Multiple sequence alignment with other GPCRs showed the presence of seven transmembrane segments and conserved motifs but interesting differences were also observed. Phylogenetic analysis grouped these new sequences within the 5-HT7 clade of GPCRs. Molecular modeling showed a striking resemblance in the spatial localization of key residues with their mammalian counterparts. Expression analysis using available RNAseq data showed that both E. granulosus sequences are expressed in larval and adult stages. Localization studies performed in E. granulosus larvae with a fluorescent probe produced a punctiform pattern concentrated in suckers. E. granulosus and M. corti larvae showed an increase in motility in response to serotonin. Heterologous expression revealed elevated levels of cAMP production in response to 5-HT and two of the GPCRs showed extremely high sensitivity to 5-HT (picomolar range). While each of these GPCRs was activated by 5-HT, they exhibit distinct pharmacological properties (5-HT sensitivity, differential responsiveness to ligands). CONCLUSIONS/SIGNIFICANCE: These data provide the first functional report of GPCRs in parasitic cestodes. The serotoninergic GPCRs characterized here may represent novel druggable targets for antiparasitic intervention.
Assuntos
Cestoides/fisiologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Animais , Cestoides/genética , Cestoides/crescimento & desenvolvimento , Infecções por Cestoides/tratamento farmacológico , Clonagem Molecular , Biologia Computacional , Echinococcus granulosus/genética , Echinococcus granulosus/fisiologia , Larva/fisiologia , Mesocestoides/genética , Mesocestoides/crescimento & desenvolvimento , Mesocestoides/fisiologia , Modelos Moleculares , Filogenia , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Serotonina/farmacologiaRESUMO
Echinococcosis is a parasitic zoonosis that is considered as a neglected disease by the World Health Organization. The species Echinococcus oligarthrus is one of the causative agents of Neotropical echinococcosis, which is a poorly understood disease that requires a complex medical examination, may threaten human life, and is frequently associated with a low socioeconomic status. Morphological and genetic diversity in E. oligarthrus remains unknown. The aim of this work is to identify and characterize E. oligarthrus infections in sylvatic animals from the Upper Paraná Atlantic Forest in the province of Misiones, Argentina, by following an integrative approach that links morphological, genetic and ecological aspects. This study demonstrates, for the first time, one of the complete life cycles of E. oligarthrus in an important ecoregion. The Upper Paraná Atlantic Forest constitutes the largest remnant continuous forest of the Atlantic Forest, representing 7% of the world's biodiversity. This is the first molecular determination of E. oligarthrus in Argentina. In addition, the agouti (Dasyprocta azarae), the ocelot (Leopardus pardalis) and the puma (Puma concolor) were identified as sylvatic hosts of Neotropical echinococcosis caused by E. oligarthrus. Mitochondrial and nuclear molecular marker analyses showed a high genetic diversity in E. oligarthrus. Moreover, the genetic distance found among E. oligarthrus isolates is higher than the one observed among Echinococcus granulosus genotypes, which clearly indicates that there are at least two different E. oligarthrus populations in Argentina. This study provides valuable information to understand the underlying conditions that favour the maintenance of E. oligarthrus in sylvatic cycles and to evaluate its zoonotic significance for devising preventive measures for human and animal wellbeing.
Assuntos
Equinococose/veterinária , Echinococcus/genética , Variação Genética , Animais , Argentina/epidemiologia , Dasyproctidae/parasitologia , Equinococose/epidemiologia , Equinococose/parasitologia , Echinococcus/classificação , Felidae/parasitologia , FilogeniaRESUMO
A molecular PCR study using DNA from 21 hydatid cysts was performed to determine which strain type is responsible for human infection in Peru. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was amplified in 20 out of 21 samples, revealing that all but 1 sample (19/20, 95%) belonged to the common sheep strain (G1). The remaining samples belonged to the camel strain (G6). The G1 genotype was most frequently found in human cases of cystic hydatid disease (CHD) in Peru. Local control measures should focus primarily on decreasing dog and sheep infection rather than intermediate reservoirs.
Assuntos
Camelus/parasitologia , DNA de Helmintos/química , DNA Mitocondrial/química , Echinococcus granulosus/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Zoonoses/parasitologia , Animais , DNA de Helmintos/análise , DNA Mitocondrial/análise , Cães , Equinococose , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Genótipo , Humanos , Peru/epidemiologia , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/parasitologia , Zoonoses/epidemiologia , Zoonoses/transmissãoRESUMO
We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Echinococcus granulosus/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , Expressão Gênica , Imuno-Histoquímica , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The detection of Echinococcus granulosus in dogs is important for epidemiological surveillance and evaluation of cystic hydatic disease control programs. We report the efficacy of two PCR-based methods to detect patent and pre-patent infection in dogs experimentally infected with E. granulosus. The detection is based on amplification of a fragment of a mitochondrial gene (Mit-PCR) and a DNA repetitive element (Rep-PCR) of E. granulosus. We tested the ability of both methods to detect several genotypes of the parasite. Both PCR methods could detect E. granulosus in pre-patent and patent periods, even when microscopical observation of eggs resulted negative in fecal samples. The Mit-PCR produced the same amplification pattern for all the parasite genotypes tested while the amplification patterns with the Rep-PCR differed among groups of strains. Fecal samples collected from dogs of an endemic area were diagnosed with more sensitivity than arecoline hydrobromide purgation. These molecular methods could be applied in the confirmation of coproantigen-positive fecal samples and to verify the success of control programs.
Assuntos
Doenças do Cão , Equinococose/diagnóstico , Echinococcus granulosus/química , Echinococcus granulosus/genética , Genótipo , Animais , DNA de Helmintos/análise , DNA Mitocondrial/análise , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Equinococose/epidemiologia , Fezes/parasitologia , Humanos , Contagem de Ovos de Parasitas , Sensibilidade e Especificidade , ZoonosesRESUMO
Echinococcus granulosus, the etiological agent of cystic hydatid disease, exists as a series of strains or genotypes, differing in biological features. Many of the secreted and membrane-bound proteins (S/M) from helminth parasites are involved in the host-parasite interplay and constitute potential targets for diagnosis, anti-parasitic drugs and vaccines. A number of E. granulosus S/M proteins were identified using the signal sequence trap technique. Six out of seven cDNA fragments of these newly identified proteins showed nucleotide and amino acid sequence variation. Inter-strain variation was reported for other characterized S/M proteins as the vaccine target EG95 and the major hydatid cyst fluid antigen, Antigen B (AgB). AgB is highly polymorphic, 101 different sequences related to AgB were reported so far and were grouped in 5 genes (EgB1-EgB5) and one pseudogene (EgB2p) exclusive of G5, G6/G7 genotypes. The significance of AgB polymorphism and possible consequences in diagnostic performance are discussed. The diagnostic value of the new protein variants detected in E. granulosus strains could be determined through standardized inter-laboratory studies as the recently done by the South American Network for Hydatid Serology.
Assuntos
Echinococcus granulosus/metabolismo , Variação Genética , Proteínas de Helminto , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/metabolismo , Echinococcus granulosus/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Dados de Sequência MolecularRESUMO
El agente causante de la hidatidosis (echinococcosis quística) en el hombre, es el cestode Echinococcus granulosus. Hasta el momento están descriptas nueve variantes genéticas: cepa oveja (genotipo G1), oveja de Tasmania (genotipo G2), cepa búfalo (genotipo G3), cepa caballo (genotipo G4), cepa vaca (genotipo G5), cepa camello (genotipo G6), cepa cerdo (genotipo G7), cepa cérvido (genotipo G8) y el genotipo G9. Estas variantes genéticas deben ser consideradas al desarrollar tareas de investigación y diagnóstico en E. granulosus y al implementar programas de control de la enfermedad. Para algunas de ellas se ha podido determinar diferencias en la infectividad para el hombre, porcentaje de quistes fértiles en diferentes hospedadores intermediarios y período prepatente en perros, entre otras. La identificación de cepas se basa en las diferencias de las características morfológicas, bioquímicas, epidemiológicas y actualmente en la caracterización del genoma usando técnicas moleculares. En la Argentina la hidatidosis es endémica, principalmente en áreas rurales, lo que permite el estudio de los patrones de transmisión, estructura genética y cepas de E. granulosus infectivas para el hombre. Hasta el momento estudios en el resto del mundo indican que la más importante fuente de infección para el hombre es la cepa oveja. Sin embargo, recientemente en Argentina se ha demostrado molecularmente que además de la cepa oveja, la cepa oveja de Tasmania y camello también son infectivas para el hombre en altas proporciones. En este trabajo multidisciplinario, llevado a cabo con personal de las áreas endémicas, el propósito fue conocer la situación epidemiológica de la hidatidosis en la Argentina, por medio de la caracterización molecular de aislamientos de E. granulosus presentes en el país. Analizar su variabilidad genética mediante regiones de ADN evolutivamente independiente, incluyendo moléculas involucradas en el diagnóstico y en la modulación de la respuesta inmune en el hombre, tratando de concentrar el análisis en aquellos aislamientos en los que hay pocos estudios realizados, como la cepa oveja de Tasmania, y la cepa camello y correlacionar dichas variantes con las caracterísitcas clínico-patológicas de la enfermedad. Los resultados obtenidos confirman la presencia de por lo menos cinco variantes genéticas: cepa oveja, oveja de Tasmania, vaca, camello y cerdo en la Argentina... (TRUNCADO) (AU)
Assuntos
Técnicas In Vitro , Equinococose/diagnóstico , Equinococose/epidemiologia , Equinococose/microbiologia , Equinococose/patologia , Echinococcus/isolamento & purificação , Echinococcus/genética , Análise de Sequência de DNA , Southern Blotting , DNA de Helmintos/isolamento & purificação , Família Multigênica , Reação em Cadeia da Polimerase , Variação Genética , Argentina/epidemiologia , Doenças Endêmicas , ZoonosesRESUMO
El agente causante de la hidatidosis (echinococcosis quística) en el hombre, es el cestode Echinococcus granulosus. Hasta el momento están descriptas nueve variantes genéticas: cepa oveja (genotipo G1), oveja de Tasmania (genotipo G2), cepa búfalo (genotipo G3), cepa caballo (genotipo G4), cepa vaca (genotipo G5), cepa camello (genotipo G6), cepa cerdo (genotipo G7), cepa cérvido (genotipo G8) y el genotipo G9. Estas variantes genéticas deben ser consideradas al desarrollar tareas de investigación y diagnóstico en E. granulosus y al implementar programas de control de la enfermedad. Para algunas de ellas se ha podido determinar diferencias en la infectividad para el hombre, porcentaje de quistes fértiles en diferentes hospedadores intermediarios y período prepatente en perros, entre otras. La identificación de cepas se basa en las diferencias de las características morfológicas, bioquímicas, epidemiológicas y actualmente en la caracterización del genoma usando técnicas moleculares. En la Argentina la hidatidosis es endémica, principalmente en áreas rurales, lo que permite el estudio de los patrones de transmisión, estructura genética y cepas de E. granulosus infectivas para el hombre. Hasta el momento estudios en el resto del mundo indican que la más importante fuente de infección para el hombre es la cepa oveja. Sin embargo, recientemente en Argentina se ha demostrado molecularmente que además de la cepa oveja, la cepa oveja de Tasmania y camello también son infectivas para el hombre en altas proporciones. En este trabajo multidisciplinario, llevado a cabo con personal de las áreas endémicas, el propósito fue conocer la situación epidemiológica de la hidatidosis en la Argentina, por medio de la caracterización molecular de aislamientos de E. granulosus presentes en el país. Analizar su variabilidad genética mediante regiones de ADN evolutivamente independiente, incluyendo moléculas involucradas en el diagnóstico y en la modulación de la respuesta inmune en el hombre, tratando de concentrar el análisis en aquellos aislamientos en los que hay pocos estudios realizados, como la cepa oveja de Tasmania, y la cepa camello y correlacionar dichas variantes con las caracterísitcas clínico-patológicas de la enfermedad. Los resultados obtenidos confirman la presencia de por lo menos cinco variantes genéticas: cepa oveja, oveja de Tasmania, vaca, camello y cerdo en la Argentina... (TRUNCADO)
Assuntos
Análise de Sequência de DNA , DNA de Helmintos/isolamento & purificação , Echinococcus/genética , Echinococcus/isolamento & purificação , Equinococose/diagnóstico , Equinococose/epidemiologia , Equinococose/microbiologia , Equinococose/patologia , Família Multigênica , Reação em Cadeia da Polimerase , Southern Blotting , Variação Genética , Argentina/epidemiologia , Doenças Endêmicas , ZoonosesRESUMO
Mitochondrial cytochrome oxidase subunit 1 (CO1) sequencing, Southern blot of a repetitive DNA element and single strand conformation polymorphism of the 5' non-transcribed region of the cytosolic malate dehydrogenase (MDH) gene were used to determine the extent and distribution of Echinococcus granulosus genetic variation in Argentina. Five distinct strains of E. granulosus were shown to exist in the country. The common sheep, Tasmanian sheep, cattle and camel strains were identified in humans. Unlike the situation found in other countries, where the common sheep strain is the major source of human contamination, the Tasmanian sheep and camel strains produced a significant number of human infections in some regions of Argentina. This is the first report of cattle strain in humans in South America. Goats could be the natural intermediate host of the camel strain, which was not identified in humans from other regions so far. More than one genotype was identified in the same geographic area. These findings may have important consequences for human health and the control of hydatid disease. Within-strain differences were also observed, showing the potential of variation of E. granulosus.