RESUMO
Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.
Assuntos
Cromatografia em Gel , Exossomos , Vesículas Extracelulares , Microscopia Eletrônica de Transmissão , Toxocara canis , Toxocara , Ultracentrifugação , Animais , Toxocara/isolamento & purificação , Toxocara/metabolismo , Toxocara/química , Toxocara canis/química , Exossomos/química , Exossomos/ultraestrutura , Exossomos/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Vesículas Extracelulares/metabolismo , Cães , Larva , Imunoprecipitação , Toxocaríase/parasitologia , Gatos , Nanopartículas/química , Tamanho da Partícula , Proteínas de Helminto/análise , Proteínas de Helminto/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificaçãoRESUMO
Schistosoma japonicum glutathione-S-transferase (SjGST), the so-called GST-tag, is one of the most widely used protein tags for the purification of recombinant proteins by affinity chromatography. Attachment of SjGST enables the purification of a protein of interest (POI) using commercially available glutathione-immobilizing resins. Here we produced an SjGST mutant pair that forms heterodimers by adjusting the salt bridge pairs in the homodimer interface of SjGST. An MD study confirmed that the SjGST mutant pair did not disrupt the heterodimer formation. The modified SjGST protein pair coexpressed in Escherichia coli was purified by glutathione-immobilized resin. The stability of the heterodimeric form of the SjGST mutant pair was further confirmed by size exclusion chromatography. Surface plasmon resonance measurements unveiled the selective formation of heterodimers within the pair, accompanied by a significant suppression of homodimerization. The heterodimeric SjGST exhibited enzymatic activity in assays employing a commercially available fluorescent substrate. By fusing one member of the heterodimeric SjGST pair with a fluorescent protein and the other with the POI, we were able to conveniently and sensitively detect protein-protein interactions using fluorescence spectroscopy in the pull-down assays. Thus, utilization of the heterodimeric SjGST would be a useful tag for protein science.
Assuntos
Cromatografia de Afinidade , Glutationa Transferase , Schistosoma japonicum , Schistosoma japonicum/enzimologia , Glutationa Transferase/metabolismo , Glutationa Transferase/química , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/genética , Animais , Cromatografia de Afinidade/métodos , Multimerização Proteica , Proteínas de Helminto/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Modelos MolecularesRESUMO
Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55 kDa. The optimal pH and temperature are found to be 8.0 and 37 °C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15 min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50 °C and 60 °C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.
Assuntos
Trematódeos , gama-Glutamiltransferase , gama-Glutamiltransferase/química , gama-Glutamiltransferase/isolamento & purificação , Trematódeos/enzimologia , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificaçãoRESUMO
The Schistosoma japonicum fatty acid-binding protein (FABP) is used in the cell membrane to absorb and transport fatty acids, which cannot be resynthesized by the organism and combined with hydrophobic ligands. Among the 5 stages of the worm life cycle examined, FABP messenger ribonucleic acid (mRNA) expression was highest in male adult worms, followed by the liver-stage schistosome, and was the lowest in the lung-stage schistosome. The fabp gene-coding region was cloned and expressed to obtain recombinant S. japonicum FABP (rSjFABP) with a molecular weight of approximately 18 kDa. Mice were then immunized against rSjFABP to prepare anti-FABP serum. Using immunohistochemical techniques, FABP protein was found to localize to the eggshell, parenchyma, and digestive tract. Double-stranded RNA-mediated knockdown of FABP mRNA by RNA interference decreased the number of transcripts by >70%. Moreover, the egg production rate decreased, whereas the abnormal egg ratio was significantly increased in the FABP-silenced group compared with the negative control group (P < 0.05). These results demonstrate that FABP localizes in adults and in various stages. FABP contributes to the egg-laying capacity of adults, which may be related to the reproductive function of S. japonicum.
Assuntos
Proteínas de Ligação a Ácido Graxo/fisiologia , Proteínas de Helminto/fisiologia , Schistosoma japonicum/fisiologia , Animais , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/imunologia , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Feminino , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Imuno-Histoquímica , Fígado/parasitologia , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma japonicum/química , Schistosoma japonicum/genéticaRESUMO
Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, and it has the potential to diminish the quality of life. Recent clinical and experimental evidence demonstrate protective aspects of parasitic helminth infection against IBD. Reports have highlighted the potential use of helminths and their byproducts as potential treatment for IBD. In the current study, we studied the effect of a newborn larvae-specific serine protease from Trichinella spiralis (TsSp) on the host immune and inflammatory responses. A 49-kDa recombinant TsSp (rTsSp) was expressed in Escherichia coli BL21 (DE3) and purified. The cytotoxicity of rTsSp was analyzed. The immune protective effect of rTsSp was studied by using dextran sodium sulfate (DSS)-induced mouse colitis model. The result illustrated that rTsSp has no toxic effects on cells. We further demonstrated that administration of the rTsSp without the additional adjuvant before the induction of DSS-induced colitis reduced the severity of intestinal inflammation and the disease index; it suppressed macrophage infiltration, reduced TNF-α secretion, and induced IL-10 expression. Our findings suggest therapeutic potential of rTsSp on colitis by altering the effect of macrophages. Data also suggest immunotherapy with rTsSp holds promise for use as an additional strategy to positively modulate inflammatory processes involved in IBD.
Assuntos
Colite/prevenção & controle , Proteínas de Helminto/química , Fatores Imunológicos/farmacologia , Serina Proteases/imunologia , Trichinella spiralis/química , Animais , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Proteínas de Helminto/isolamento & purificação , Interleucina-10/metabolismo , Larva/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Trichinella spiralis/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The flatworm Taenia solium causes human and pig cysticercosis. When cysticerci are established in the human central nervous system, they cause neurocysticercosis, a potentially fatal disease. Neurocysticercosis is a persisting public health problem in rural regions of Mexico and other developing countries of Latin America, Asia, and Africa, where the infection is endemic. The great variability observed in the phenotypic and genotypic traits of cysticerci result in a great heterogeneity in the patterns of molecules secreted by them within their host. This work is aimed to identify and characterize cysticercal secretion proteins of T. solium cysticerci obtained from 5 naturally infected pigs from Guerrero, Mexico, using 2D-PAGE proteomic analysis. The isoelectric point (IP) and molecular weight (MW) of the spots were identified using the software ImageMaster 2D Platinum v.7.0. Since most secreted proteins are impossible to identify by mass spectrometry (MS) due to their low concentration in the sample, a novel strategy to predict their sequence was applied. In total, 108 conserved and 186 differential proteins were identified in five cysticercus cultures. Interestingly, we predicted the sequence of 14 proteins that were common in four out of five cysticercus cultures, which could be used to design vaccines or diagnostic methods for neurocysticercosis. A functional characterization of all sequences was performed using the algorithms SecretomeP, SignalP, and BlastKOALA. We found a possible link between signal transduction pathways in parasite cells and human cancer due to deregulation in signal transduction pathways. Bioinformatics analysis also demonstrated that the parasite release proteins by an exosome-like mechanism, which could be of biological interest.
Assuntos
Cysticercus/metabolismo , Proteoma , Taenia solium/metabolismo , Animais , Cisticercose/veterinária , Eletroforese em Gel Bidimensional , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Análise de Sequência de Proteína , Transdução de Sinais , Suínos , Doenças dos Suínos/parasitologia , Taenia solium/genética , Taenia solium/crescimento & desenvolvimentoRESUMO
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
Tolerogenic dendritic cells (tolDCs) are central players in the maintenance of immune tolerance and thereby have been identified as the most favourable candidates for cell therapy of autoimmune diseases. We have recently shown that excretory-secretory products (ES L1) released by Trichinella spiralis larvae induce stable human tolDCs in vitro via Toll-like receptor 2 (TLR2) and TLR4. However, engagement of these receptors did not fully explain the tolerogenic profile of DCs. Here, we observed for the first time that dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) interacts with highly glycosylated ES L1 and contributes to the generation of ES L1-induced tolDCs. Blocking DC-SIGN interfered with the ES L1-induced higher expression of CD40 and CCR7 and the production of IL-10 and TGF-ß by DCs. The cooperation of TLR2, TLR4 and DC-SIGN receptors is of importance for the capacity of DCs to prime T cell response toward Th2 and to induce expansion of CD4+CD25+Foxp3+ T cells, as well as for the production of IL-10 and TGF-ß by these cells. Overall, these results indicate that induction of tolDCs by ES L1 involves engagement of multiple pattern recognition receptors namely, TLR2, TLR4 and DC-SIGN.
Assuntos
Antígenos de Helmintos/farmacologia , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Trichinella spiralis/imunologia , Animais , Antígenos de Helmintos/isolamento & purificação , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Voluntários Saudáveis , Proteínas de Helminto/isolamento & purificação , Humanos , Larva/imunologia , Larva/metabolismo , Masculino , Cultura Primária de Células , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.
Assuntos
Angiostrongylus cantonensis/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Sequência de Aminoácidos , Angiostrongylus cantonensis/química , Animais , Antígenos de Helmintos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Sequência Conservada , Reações Cruzadas , Eletroforese , Eletroforese em Gel Bidimensional , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Humanos , Imunoensaio , Testes Imunológicos , Espectrometria de Massas , Meningoencefalite/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.
Assuntos
Proteínas de Helminto , Proteínas Recombinantes , Schistosoma japonicum , Animais , Anticorpos Anti-Helmínticos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismoRESUMO
Schistosomiasis is one of the most important helminthic parasitic infections in the world, with over 700 million people at risk of infection. Species of Schistosoma have a complex life cycle involving the infection of freshwater snails before infecting their mammalian definitive host. Taking about 130,000 lives per annum, S. mansoni is the major cause of intestinal schistosomiasis worldwide. Within Biomphalaria glabrata snails, asexual replication of the parasite gives rise to cercariae larvae. Cercariae actively penetrate the host's skin to complete their life cycle and eventually transform into adult worms. If left untreated, intestinal schistosomiasis can lead to peripheral destruction of the portal vein system, gastric hemorrhage from esophageal varices, as well as hepatic failure. Mass spectrometry (MS) is the method of choice for proteomics analysis. The bottom-up proteomics approach-also known as "shotgun proteomics"-typically includes a protein extraction and solubilization step followed by proteolytic digestion and tandem MS (MS/MS) analysis. Proteins are later identified by peptide de novo sequencing upon MS and MS/MS spectra of digest peptides. In this chapter, we introduce an analytical workflow for proteome profiling of S. mansoni cercariae using bottom-up proteomics. The cercariae were isolated and lysed. Proteins were then extracted, enzymatically digested, and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins were identified using MaxQuant software. Cercariae are the first life stage of the parasite S. mansoni which humans encounter, and conducting proteomic analysis on this life cycle stage can shed light on possible drug or vaccine candidates to help disable the parasite's ability to infect or arm the immune system for parasite clearance.
Assuntos
Cercárias/metabolismo , Proteínas de Helminto/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Schistosoma mansoni/metabolismo , Animais , Biomphalaria/parasitologia , Cromatografia Líquida , Proteínas de Helminto/isolamento & purificação , Parasitos/metabolismo , Espectrometria de Massas em TandemRESUMO
Proteins secreted by, or displayed on the surface tegument of, trematodes have key functions in the host-parasite interaction. As such, they are often leading targets for diagnostic tests or vaccine candidates. Here we describe methods for the isolation and analysis of soluble secreted proteins (i.e., the secretome) released during in vitro culture of adult Fasciola hepatica. We also describe two methods for the enrichment of proteins displayed on the outer tegumental surface of F. hepatica. These approaches enable downstream identification of the isolated proteins by mass spectrometry-based proteomics.
Assuntos
Fasciola hepatica/química , Proteínas de Helminto/isolamento & purificação , Espectrometria de Massas/métodos , Proteínas de Membrana/isolamento & purificação , Proteômica/métodos , Animais , Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Membrana/metabolismoRESUMO
This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.
Assuntos
Fasciola hepatica/química , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Macrófagos/parasitologia , Animais , Fasciolíase/parasitologia , Humanos , Focalização Isoelétrica/métodos , Leucócitos Mononucleares/parasitologia , Monócitos/parasitologiaRESUMO
The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.
Assuntos
Acetilcolina/metabolismo , Canais de Cloreto/química , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/química , Haemonchus/metabolismo , Proteínas de Helminto/química , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Aldicarb/farmacologia , Sequência de Aminoácidos , Animais , Anti-Helmínticos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Carbacol/metabolismo , Carbacol/farmacologia , Canais de Cloreto/genética , Canais de Cloreto/isolamento & purificação , Canais de Cloreto/metabolismo , Colina/análogos & derivados , Colina/metabolismo , Colina/farmacologia , Clonagem Molecular , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/isolamento & purificação , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemonchus/genética , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/metabolismo , Modelos Moleculares , Oócitos/citologia , Oócitos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.
Assuntos
Expressão Gênica , Proteínas de Helminto/genética , Proteínas Recombinantes/genética , Trichinella/genética , Leveduras/genética , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Citocinas/metabolismo , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/metabolismo , Camundongos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Trichinella/imunologia , Trichinella/metabolismo , Leveduras/metabolismoRESUMO
Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
Assuntos
Anexinas/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Sequência de Aminoácidos/genética , Animais , Anexinas/química , Anexinas/genética , Anexinas/isolamento & purificação , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Sequência de Bases/genética , Dicroísmo Circular , Biologia Computacional , Modelos Animais de Doenças , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Humanos , Masculino , Camundongos , Microscopia Imunoeletrônica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/sangue , Esquistossomose mansoni/parasitologia , Relação Estrutura-AtividadeRESUMO
Fasciola hepatica is a globally distributed zoonotic trematode that causes fasciolosis in livestock, wildlife, ruminants and humans. Fasciolosis causes a significant economic impact on the agricultural sector and affects human health. Due to the increasing prevalence of triclabendazole resistance in F. hepatica, alternative treatment methods are required. Many protein antigens have been trialled as vaccine candidates with low success, however, the tegument of F. hepatica is highly glycosylated and the parasite-derived glycoconjugate molecules have been identified as an important mediator in host-parasite interactions and as prime targets for the host immune system. Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are two ruminant-specific glycan-binding proteins, showing upregulation in the bile duct of sheep infected with F. hepatica, which are believed to mediate host-parasite interaction and innate immunity against internal parasites. For the first known time, this study presents the ligand profile of whole worm and tegument extracts of F. hepatica that interacted with immobilised LGALS-11 and LGALS-14. LGALS-14 interacted with a total of 255 F. hepatica proteins. The protein which had the greatest interaction was identified as an uncharacterised protein which contained a C-type lectin domain. Many of the other proteins identified were previously trialled vaccine candidates including glutathione S-transferase, paramyosin, cathepsin L, cathepsin B, fatty acid binding protein and leucine aminopeptidase. In comparison to LGALS-14, LGALS-11 interacted with only 49 F. hepatica proteins and it appears to have a much smaller number of binding partners in F. hepatica. This is, to our knowledge, the first time host-specific lectins have been used for the enrichment of F. hepatica glycoproteins and this study has identified a number of glycoproteins that play critical roles in host-parasite interactions which have the potential to be novel vaccine candidates.
Assuntos
Antígenos de Helmintos/análise , Fasciola hepatica/crescimento & desenvolvimento , Galectinas/metabolismo , Proteínas de Helminto/análise , Interações Hospedeiro-Parasita , Mapeamento de Interação de Proteínas , Ovinos , Animais , Antígenos de Helmintos/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Ligantes , Espectrometria de Massas , Ligação Proteica , ProteômicaRESUMO
BACKGROUND: Gnathostoma spinigerum is a harmful parasitic nematode that causes severe morbidity and mortality in humans and animals. Effective drugs and vaccines and reliable diagnostic methods are needed to prevent and control the associated diseases; however, the lack of genome, transcriptome, and proteome databases remains a major limitation. In this study, transcriptomic and secretomic analyses of advanced third-stage larvae of G. spinigerum (aL3Gs) were performed using next-generation sequencing, bioinformatics, and proteomics. RESULTS: An analysis that incorporated transcriptome and bioinformatics data to predict excretory-secretory proteins (ESPs) classified 171 and 292 proteins into classical and non-classical secretory groups, respectively. Proteins with proteolytic (metalloprotease), cell signaling regulatory (i.e., kinases and phosphatase), and metabolic regulatory function (i.e., glucose and lipid metabolism) were significantly upregulated in the transcriptome and secretome. A two-dimensional (2D) immunomic analysis of aL3Gs-ESPs with G. spinigerum-infected human sera and related helminthiases suggested that the serine protease inhibitor (serpin) was a promising antigenic target for the further development of gnathostomiasis immunodiagnostic methods. CONCLUSIONS: The transcriptome and excretory-secretory proteome of aL3Gs can facilitate an understanding of the basic molecular biology of the parasite and identifying multiple associated factors, possibly promoting the discovery of novel drugs and vaccines. The 2D-immunomic analysis identified serpin, a protein secreted from aL3Gs, as an interesting candidate for immunodiagnosis that warrants immediate evaluation and validation.
Assuntos
Gnathostoma/genética , Proteínas de Helminto/genética , Testes Imunológicos , Larva/genética , Proteoma , Transcriptoma , Animais , Antígenos de Helmintos/genética , Biologia Computacional/métodos , Gnatostomíase/tratamento farmacológico , Proteínas de Helminto/isolamento & purificação , HumanosRESUMO
Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.
Assuntos
Búfalos/sangue , Cromatografia Líquida , Fasciola/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Espectrometria de Massas em Tandem , Animais , Búfalos/parasitologia , Fasciola/química , Fasciola/imunologia , Fasciola hepatica/imunologia , Fasciolíase/imunologia , Fasciolíase/parasitologia , Proteínas de Helminto/isolamento & purificação , Interações Hospedeiro-Parasita , ProteômicaRESUMO
Antimicrobial peptides from marine invertebrates are known not only to act like cytotoxic agents, but they also can display some additional activities in mammalian organisms. In particular, these peptides can modulate the complement system as was described for tachyplesin, a peptide from the horseshoe crab. In this work, we investigated the influence on complement activation of the antimicrobial peptide arenicin-1 from the marine polychaete Arenicola marina. To study effects of arenicin on complement activation in human blood serum, we used hemolytic assays of two types, with antibody sensitized sheep erythrocytes and rabbit erythrocytes. Complement activation was also assessed, by the level of C3a production that was measured by ELISA. We found that the effect of arenicin depends on its concentration. At relatively low concentrations the peptide stimulates complement activation and lysis of target erythrocytes, whereas at higher concentrations arenicin acts as a complement inhibitor. A hypothetical mechanism of peptide action is proposed, suggesting its interaction with two complement proteins, C1q and C3. The results lead to the possibility of the development of new approaches for therapy of diseases connected with complement dysregulation, using peptide regulators derived from natural antimicrobial peptides of invertebrates.