RESUMO
Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.
Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.
Assuntos
Sequência de Aminoácidos , Catepsina L , Echinococcus , Filogenia , Animais , Catepsina L/genética , Echinococcus/enzimologia , Echinococcus/genética , Echinococcus/classificação , Alinhamento de Sequência , Clonagem Molecular , Proteínas de Helminto/genética , Proteínas de Helminto/química , Estágios do Ciclo de Vida , Equinococose/parasitologia , Domínio Catalítico , Perfilação da Expressão GênicaRESUMO
Septins are cytoskeletal proteins and their interaction with membranes is crucial for their role in various cellular processes. Septins have polybasic regions (PB1 and PB2) which are important for lipid interaction. Earlier, we and others have highlighted the role of the septin C-terminal domain (CTD) to membrane interaction. However, detailed information on residues/group of residues important for such feature is lacking. In this study, we investigate the lipid-binding profile of Schistosoma mansoni Septin10 (SmSEPT10) using PIP strip and Langmuir monolayer adsorption assays. Our findings highlight the CTD as the primary domain responsible for lipid interaction in SmSEPT10, showing binding to phosphatidylinositol phosphates. SmSEPT10 CTD contains a conserved polybasic region (PB3) present in both animals and fungi septins, and a Lys (K367) within its putative amphipathic helix (AH) that we demonstrate as important for lipid binding. PB3 deletion or mutation of this Lys (K367A) strongly impairs lipid interaction. Remarkably, we observe that the AH within a construct lacking the final 43 amino acid residues is insufficient for lipid binding. Furthermore, we investigate the homocomplex formed by SmSEPT10 CTD in solution by cross-linking experiments, CD spectroscopy, SEC-MALS and SEC-SAXS. Taken together, our studies define the lipid-binding region in SmSEPT10 and offer insights into the molecular basis of septin-membrane binding. This information is particularly relevant for less-studied non-human septins, such as SmSEPT10.
Assuntos
Schistosoma mansoni , Septinas , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Septinas/metabolismo , Septinas/química , Septinas/genética , Animais , Ligação Proteica , Domínios Proteicos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Lipídeos/químicaRESUMO
Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.
Assuntos
Proliferação de Células , Hidroxiureia , Taenia , Hidroxiureia/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Taenia/efeitos dos fármacos , Taenia/genética , Taenia/crescimento & desenvolvimento , Taenia/metabolismo , Proteômica/métodos , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteoma/metabolismo , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismoRESUMO
Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of â¼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.
Assuntos
Echinococcus granulosus , Proteínas de Helminto , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Animais , Cães , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/química , Bovinos , Sequência de Aminoácidos , Tripsina/química , Tripsina/metabolismoRESUMO
Nuclear receptors are ligand-activated transcription factors capable of regulating the expression of complex gene networks. The family includes seven subfamilies of protein with a wide phylogenetic distribution. A novel subfamily with two DNA-binding domains (2DBDs) has been first reported in Schistosoma mansoni (Platyhelminth, Trematoda). Employing an ab initio protocol and homology modeling methods, the full-length 3D structure of the Eg2DBDα.1 nuclear receptor from Echinococcus granulosus (Platyhelminth, Cestoda) was generated. The model analysis reveals the presence of the conserved three-layered alpha-helical sandwich structure in the ligand binding domain, and a particularly long and flexible hinge region. Molecular dynamics simulations were performed previous to dock a conformational library of fatty acids and retinoic acids. Our results indicate that oleic and linoleic acids are suitable ligands to this receptor. The ligand-protein complex is stabilized mainly by hydrogen bonds and hydrophobic interactions. The fact that 2DBD nuclear receptors have not been identified in vertebrates confers particular interest to these nuclear receptors, not only concerning their structure and function but as targets of new anthelmintic drugs.Communicated by Ramaswamy H. Sarma.
Assuntos
Echinococcus granulosus , Animais , Echinococcus granulosus/metabolismo , Simulação de Dinâmica Molecular , Filogenia , Ligantes , Proteínas de Helminto/química , Receptores Citoplasmáticos e Nucleares , Simulação de Acoplamento MolecularRESUMO
INTRODUCTION: Schistosomes are long-lived blood dwelling helminth parasites using intricate mechanisms to invade, mature, and reproduce inside their vertebrate hosts, whilst simultaneously deploying immune evasion strategies. Their multi-tissue organization and solid body plan presents particular problems for the definition of sub-proteomes. AREAS COVERED: Here, we focus on the two host-parasite interfaces of the adult worm accessible to the immune system, namely the tegument and the alimentary tract, but also on the secretions of the infective cercaria, the migrating schistosomulum and the mature egg. In parallel, we introduce the concepts of "leakyome' and 'disintegrome' to emphasize the importance of interpreting data in the context of schistosome biology so that misleading conclusions about the distinct proteome compositions are avoided. Lastly, we highlight the possible clinical implications of the reviewed proteomic findings for pathogenesis, vaccine design and diagnostics. EXPERT OPINION: Proteomics has provided considerable insights into the biology of schistosomes, most importantly for rational selection of novel vaccine candidates that might confer protective immunity, but also into the pathogenesis of schistosomiasis. However, given the increasing sensitivity of mass spectrometric instrumentation, we stress the need for care in data interpretation since schistosomes do not deviate from the fundamental rules of eukaryotic cell biology.
Assuntos
Esquistossomose , Vacinas , Animais , Proteômica/métodos , Proteínas de Helminto , Schistosoma , Esquistossomose/parasitologia , Esquistossomose/prevenção & controle , Proteoma/genéticaRESUMO
Cystic echinococcosis is a zoonotic disease caused by the larval stage of Echinococcus granulosus. This affliction is an endemic worldwide condition that represents a neglected parasitic disease with important socioeconomic repercussions. Proteomic characterization of larval and adult stages of E. granulosus, as well as the association between expression profiles and host interactions, is relevant for a better understanding of parasite biology, and eventually for drug design and vaccine development. This study aimed to develop a synthesis of the evidence available related to proteomics of E. granulosus. A systematic review was carried out to collect data concerning the proteomics of E. granulosus, without language or host restriction, published between 1980 and 2019. A systematic search was carried out in the Trip Database, BIREME-BVS, SciELO, Web of Science, PubMed, EMBASE, SCOPUS, EBSCO host, and LILACS, using MeSH terms, free words, and Boolean connectors, and adapting strategies to each source of information. Additionally, a manual cross-reference search was performed. Variables studied were the year of publication, geographic origin of the study, number of samples, hosts, parasitic organs, proteomic techniques, and parasite proteins verified. Nine-hundred and thirty-six related articles were identified: 17 fulfilled selection criteria, including slightly more than 188 samples. Most articles were published between 2014 and 2019 (64.7%) and were from Brazil and China (35.3% each). In reference to confirmed hosts in the primary articles, cattle (41.2%) and humans (23.5%) were the most frequently reported. Concerning proteomic techniques applied in the primary articles, LC-MS/MS was the most used (41.1%), and 890 proteins were reported by the primary articles. As the results of our search suggest, the information related to E. granulosus proteomics is scarce, heterogeneous, and scattered throughout several articles that include a diversity of tissues, samples, intermediate hosts, and proteomic techniques. Consequently, the level of evidence generated by our search is type 4.
Assuntos
Equinococose/parasitologia , Echinococcus granulosus/química , Proteínas de Helminto/análise , Proteômica , Animais , Proteínas de Helminto/químicaRESUMO
Parastrigea brasiliana (Szidat, 1928) Dubois, 1964, was described from (Cochlearius cochlearius) in South America. The taxonomy of this species has been unstable due that it was described as a member of Strigea Abildgaard, 1790. However, the same author one year later transferred it to Apharyngostrigea Ciurea, 1927 and since then, it has been alternatively placed in the genus Apharyngostrigea or Parastrigea Szidat, 1928 from Strigeidae. In the current research, specimens identified as P. brasiliana were collected from type host in southeastern Mexico. We sequenced three molecular markers: the internal transcribed spacers ITS1 and ITS2 including the 5.8S gene (ITS region), the D1-D3 domains of the large subunit (LSU) from nuclear DNA and cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA. These sequences were aligned with other sequences available in the GenBank dataset from Strigeidae. Maximum likelihood and Bayesian analyses inferred with three molecular markers consistently showed that P. brasiliana is not closely related to other members of the genus Parastrigea and are placed in a reciprocal monophyletic clade inside Apharyngostrigea, with very low genetic divergence, varying from 0 to 0.09% for the ITS, from 0 to 0.08% for the LSU and from 0.21 to 0.43% for cox 1. Consequently, we proposed to reallocate it to A. brasiliana. The phylogenetic analyses obtained are key and very useful for re-evaluate the morphology of A. brasiliana because this species share morphological characters with the genera Parastrigea (concentration of vitelline follicles distributed in two lateral expansions on the forebody) and Apharyngostrigea (absence of pharynx). Finally, the current record of A. brasiliana expands its distribution range in four countries, namely, the USA, Mexico, Venezuela and Brazil, in the Neotropical region.
Assuntos
Doenças das Aves/parasitologia , Aves , Trematódeos , Infecções por Trematódeos/veterinária , Animais , DNA de Helmintos/análise , DNA Mitocondrial/análise , Proteínas de Helminto/análise , México , Microscopia Eletrônica de Varredura/veterinária , Trematódeos/anatomia & histologia , Trematódeos/classificação , Trematódeos/genética , Trematódeos/ultraestrutura , Infecções por Trematódeos/parasitologiaRESUMO
Due to their phylogenetic proximity to humans, nonhuman primates (NHPs) are considered an adequate choice for a basic and preclinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of Toll-like receptor 4 (TLR4) suppressing the LPS-induced proinflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by intravenous (i.v.) infusion with lethal doses of live Escherichia coli. Fh15 was administered as an isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, Fh15 (i) significantly prevented bacteremia, suppressed LPS levels in plasma, and the production of C-reactive protein and procalcitonin, which are key signatures of inflammation and bacterial infection, respectively; (ii) reduced the production of proinflammatory cytokines; and (iii) increased innate immune cell populations in blood, which suggests a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This report is the first to demonstrate that a F. hepatica-derived molecule possesses potential as an anti-inflammatory drug against sepsis in an NHP model. IMPORTANCE Sepsis caused by Gram-negative bacteria affects 1.7 million adults annually in the United States and is one of the most important causes of death at intensive care units. Although the effective use of antibiotics has resulted in improved prognosis of sepsis, the pathological and deathly effects have been attributed to the persistent inflammatory cascade. There is a present need to develop anti-inflammatory agents that can suppress or neutralize the inflammatory responses and prevent the lethal consequences of sepsis. We demonstrated here that a small molecule of 14.5 kDa can suppress the bacteremia, endotoxemia, and many other inflammatory markers in an acute Gram-negative sepsis rhesus macaque model. These results reinforce the notion that Fh15 constitutes an excellent candidate for drug development against sepsis.
Assuntos
Anti-Inflamatórios/administração & dosagem , Bacteriemia/tratamento farmacológico , Fasciola hepatica/metabolismo , Proteínas de Ligação a Ácido Graxo/administração & dosagem , Bactérias Gram-Negativas/fisiologia , Proteínas de Helminto/administração & dosagem , Animais , Anti-Inflamatórios/metabolismo , Bacteriemia/genética , Bacteriemia/imunologia , Bacteriemia/microbiologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Fasciola hepatica/química , Fasciola hepatica/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Humanos , Macaca mulatta , Masculino , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
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
Lymphatic filariasis (LF) is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi, or Brugia timori. It is a tropical and subtropical illness that affects approximately 67 million people worldwide and that still requires better diagnostic tools to prevent its spread and enhance the effectiveness of control procedures. Traditional parasitological tests and diagnostic methods based on whole protein extracts from different worms are known for problems related to sample time collection, sensitivity, and specificity. More recently, new diagnostic tools based on immunological methods using recombinant antigens have been developed. The current review describes the several recombinant antigens used as tools for lymphatic filariasis diagnosis in antigen and antibody capture assays, highlighting their advantages and limitations as well as the main commercial tests developed based on them. The literature chronology is from 1991 to 2021. First, it describes the historical background related to the identification of relevant antigens and the generation of the recombinant polypeptides used for the LF diagnosis, also detailing features specific to each antigen. The subsequent section then discusses the use of those proteins to develop antigen and antibody capture tests to detect LF. So far, studies focusing on antibody capture assays are based on 13 different antigens with at least six commercially available tests, with five proteins further used for the development of antigen capture tests. Five antigens explored in this paper belong to the SXP/RAL-2 family (BmSXP, Bm14, WbSXP-1, Wb14, WbL), and the others are BmShp-1, Bm33, BmR1, BmVAH, WbVAH, BmALT-1, BmALT-2, and Wb123. It is expected that advances in research with these antigens will allow further development of tests combining both sensitivity and specificity with low costs, assisting the Global Program to Eliminate Lymphatic Filariasis (GPELF).
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Filariose Linfática/diagnóstico , Filariose Linfática/parasitologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/classificação , Brugia/química , Brugia/imunologia , Filariose Linfática/classificação , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Humanos , Imunoglobulina G/imunologia , Sensibilidade e Especificidade , Wuchereria bancrofti/química , Wuchereria bancrofti/imunologiaRESUMO
The present study describes three new species of monogenean parasites of characid fishes from the Upper Paraná River basin, Brazil: Characithecium paranapanemense n. sp. on Psalidodon paranae and Psalidodon bockmanni, Diaphorocleidus magnus n. sp. on Astyanax lacustris and Psalidodon fasciatus, and Diaphorocleidus neotropicalis n. sp. on Astyanax lacustris and P. bockmanni. An amendment for Diaphorocleidus is proposed, since additional characters observed in the new species required to extend the generic diagnostic features mainly to include: articulation process connecting the base of the MCO with accessory piece present or absent, and accessory piece with variable shapes (plate-like, pincer-shaped, wrench-shaped, sheath-shaped), divided or not into subunits. Characithecium paranapanemense n. sp. can be distinguished from other congeners by the morphology of its MCO and accessory piece. Diaphorocleidus magnus n. sp. differs from most of its congeners by the morphology of its accessory piece, the presence of articulation process connecting the base of the MCO with accessory piece, and the morphology of the sclerotized structures of the haptor. Diaphorocleidus neotropicalis n. sp. can be easily distinguished from its congeners by the morphology of the accessory piece, the sclerotized structures of the haptor and the morphology of the vagina. Molecular data of the new species (partial 28S rDNA and mitochondrial cytochrome oxidase I) were obtained and the first phylogenetic analysis based on 28S rDNA gene sequences for species of Characithecium and Diaphorocleidus are provided. Although Diaphorocleidus and Characithecium share some morphological similarities, phylogenetic analysis indicates that species of these two genera are not closely related.
Assuntos
Characidae , Doenças dos Peixes/parasitologia , Trematódeos/classificação , Infecções por Trematódeos/veterinária , Animais , Brasil/epidemiologia , DNA de Helmintos/análise , DNA Ribossômico/análise , Complexo IV da Cadeia de Transporte de Elétrons/análise , Proteínas de Helminto/análise , Masculino , Proteínas Mitocondriais/análise , Prevalência , Trematódeos/anatomia & histologia , Trematódeos/citologia , Trematódeos/genética , Infecções por Trematódeos/parasitologiaRESUMO
The Excretory/Secretory (ES) proteins of parasites are involved in invasion and colonization of their hosts. In addition, since ES proteins circulate in the extracellular space, they can be more accessible to drugs than other proteins, which makes ES proteins optimal targets for the development of new and better pharmacological strategies. Monogeneans are a group of parasitic Platyhelminthes that includes some pathogenic species problematic for finfish aquaculture. In the present study, 8297 putative ES proteins from four monogenean species which genomic resources are publicly available were identified and functionally annotated by bioinformatic tools. Additionally, for comparative purposes, ES proteins in other parasitic and free-living platyhelminths were identified. Based on data from the monogenean Gyrodactylus salaris, 15 ES proteins are considered potential drug targets. One of them showed homology to 10 cathepsins with known 3D structure. A docking molecular analysis uncovered that the anthelmintic emodepside shows good affinity to these cathepsins suggesting that emodepside can be experimentally tested as a monogenean's cathepsin inhibitor.
Assuntos
Antiplatelmínticos/química , Biologia Computacional , Desenvolvimento de Medicamentos , Proteínas de Helminto/genética , Trematódeos/efeitos dos fármacos , AnimaisRESUMO
Acute schistosomiasis (AS) manifests with a broad spectrum of clinical features in pediatric populations. Diagnosis may be difficult in the absence of detectable numbers of eggs. As a result, new approaches may be required to achieve an accurate diagnosis. Optimal praziquantel (PZQ) treatment regimen for young children is debatable. Also, the post-treatment response is still poorly evaluated due to the lack of reliable markers. A group of 6 children (a toddler and 5 pre-school children) and one pre-adolescent were investigated for AS clinical manifestations and followed-up for two years after treatment. Ova detection was performed by Kato-Katz (KK) and presence of Schistosoma mansoni DNA was assessed by real-time PCR (rt-PCR) in stool samples. IgG and IgE anti-Schistosoma levels and urinary antigen were detected by ELISA and point-of-care circulating cathodic antigen (POC-CCA) testing in serum and urine, respectively. AS clinical symptoms were present in 5/7 (71.4%) of the infected children, and hypereosinophilia was detected in all of them. Ova detection and serology were positive in only 3/7 (44.9%) and 4/7 (57.1%), respectively. However, real-time PCR (rt-PCR) showed the presence of Schistosoma DNA in 6/7 (85.7%) of the cases, and urinary antigen was detected in all infected children. The long-term follow-up after treatment with three doses of PZQ (80mg/kg/dose), showed high cure rates (CR) as demonstrated by the DNA-based assay as well as reduced levels of side effects. CR based on urinary antigen detection ranged from 28.6 to 100%, being the highest CR due to double testing the 2-year post-treatment samples. The results suggest that high dose and repeated treatment with PZQ might be effective for AS in young children. Also, new laboratory markers should be considered to diagnosis and monitor the drug response.
Assuntos
Anti-Helmínticos/uso terapêutico , Parasitologia , Praziquantel/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/tratamento farmacológico , Adolescente , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/urina , Biomarcadores/sangue , Biomarcadores/urina , Pré-Escolar , DNA de Helmintos/genética , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Glicoproteínas/urina , Proteínas de Helminto/urina , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Lactente , Masculino , Contagem de Ovos de Parasitas , Testes Imediatos , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/parasitologia , Testes Sorológicos , Resultado do TratamentoRESUMO
A case of abdominal dioctophymosis in a domestic cat was found in San Juan Bautista district, the Peruvian rainforest, in the Loreto department of Peru. The pet went to a veterinary clinic for a routine ovariohysterectomy during which a large nematode was found in the abdominal cavity. The nematode was morphologically identified as an adult female of Dioctophyme sp. A few morphological parameters, such as the vagina distance from the anterior part and the egg size, were different than D. renale. Partial sequences of the cytochrome c oxidase subunit I (cox1) and the small subunit 18S ribosomal RNA genes were compared with the references from public sequence database and showed a genetic identifies of 89.25% and 99.65% with D. renale, respectively. This is the first mitochondrial molecular analysis of a Dioctophyme specimen from South America and the results showed up to 12.5% nucleotide sequence variation in cox 1 gene of D. renale.
Assuntos
Doenças do Gato/parasitologia , Dioctophymatoidea/isolamento & purificação , Infecções por Enoplida/veterinária , Infecções Intra-Abdominais/veterinária , Animais , Doenças do Gato/diagnóstico , Gatos , Ciclo-Oxigenase 1/análise , Dioctophymatoidea/classificação , Infecções por Enoplida/diagnóstico , Infecções por Enoplida/parasitologia , Feminino , Proteínas de Helminto/análise , Infecções Intra-Abdominais/diagnóstico , Infecções Intra-Abdominais/parasitologia , Peru , RNA de Helmintos/análise , RNA Ribossômico 18S/análise , Floresta Úmida , Análise de Sequência de DNA/veterinária , Análise de Sequência de RNA/veterináriaRESUMO
The enzyme Glyceraldehyde-3-Phosphate Dehydrogenase from Schistosoma mansoni (SmGAPDH) is characterized as a therapeutical target for schistosomiasis. In this context, we report here the experimental structure, structural analyses and comparisons of SmGAPDH, the first one from a Platyhelminth. The enzyme was expressed, purified and assayed for crystallization, what allowed the obtainment of crystals of sufficient quality to collect X-ray diffraction data up to 2.51 Å resolution. SmGAPDH is the only GAPDH to present the sequence NNR (its residues 114-116) which leads to (especially R116) a hydrogen bond network that possibly reflects on the flexibility of residues to interact with the adenine part of NAD+, speculated to be important for differential drug design.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Proteínas de Helminto/química , Modelos Moleculares , Schistosoma mansoni/enzimologia , Animais , Cristalografia por Raios XRESUMO
Most parasitic flatworms go through different life stages with important physiological and morphological changes. In this work, we used a transcriptomic approach to analyze the main life-stages of the model tapeworm Hymenolepis microstoma (eggs, cysticercoids, and adults). Our results showed massive transcriptomic changes in this life cycle, including key gene families that contribute substantially to the expression load in each stage. In particular, different members of the cestode-specific hydrophobic ligand-binding protein (HLBP) family are among the most highly expressed genes in each life stage. We also found the transcriptomic signature of major metabolic changes during the transition from cysticercoids to adult worms. Thus, this work contributes to uncovering the gene expression changes that accompany the development of this important cestode model species, and to the best of our knowledge represents the first transcriptomic study with robust replicates spanning all of the main life stages of a tapeworm.
Assuntos
Hymenolepis/genética , Estágios do Ciclo de Vida , Transcriptoma , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Hymenolepis/crescimento & desenvolvimento , Hymenolepis/metabolismo , Família MultigênicaRESUMO
The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.
Assuntos
Echinococcus granulosus/química , Proteínas de Helminto/farmacologia , Interleucina-12/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Helminto/isolamento & purificação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
It is possible to gain a deeper insight into the role of water in biology by using physicochemical variant molecules, such as deuterium oxide (D2 O); however, D2 O is toxic to multicellular organisms in high concentrations. By using a unique desiccation-rehydration process, we demonstrate that the anhydrobiotic nematode Panagrolaimus superbus is able to tolerate and proliferate in 99 % D2 O. Moreover, we analysed P. superbus' water-channel protein (aquaporin; AQP), which is associated with dehydration/rehydration, by comparing its primary structure and modelling its tertiary structure in silico. Our data evidence that P. superbus' AQP is an aquaglyceroporin, a class of water channel known to display a wider pore; this helps to explain the rapid and successful organismal influx of D2 O into this species. This is the first demonstration of an animal able to withstand high D2 O levels, thus paving a way for the investigation of the effects D2 O on higher levels of biological organization.
Assuntos
Óxido de Deutério/metabolismo , Nematoides/metabolismo , Sequência de Aminoácidos , Animais , Aquaporinas/química , Aquaporinas/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Humanos , Nematoides/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4.