RESUMO
Neglected tropical diseases (NTDs) share certain traits: they are parasitic infections, prevailing in tropical environments and affecting marginalized sectors of the population. Six NTDs - ascariasis, cysticercosis, echinococcosis, hookworm infection, onchocerciasis and trichuriasis - all of them endemic in Latin America and the Caribbean (LAC), are analysed in this work. This review aims to discuss key information on the function of excretory/secretory (E/S) proteins from these parasites in their infectivity, pathogeny and diagnosis. The modulation of the host immune system to favour the permanence and survival of the parasite is also discussed. An updated knowledge on the function of E/S molecules in endemic parasitoses in LAC may lead to new approaches for the clinical management and diagnosis of these diseases. In turn, this could allow us to optimize their treatment and make it more affordable - a relevant goal given the economic constraints that the region is facing.
Assuntos
Doenças Endêmicas , Proteínas de Helminto/fisiologia , Helmintíase/epidemiologia , Sistema Imunitário/parasitologia , Doenças Negligenciadas/parasitologia , Animais , Região do Caribe/epidemiologia , Gerenciamento Clínico , Helmintíase/imunologia , Helmintíase/parasitologia , Interações Hospedeiro-Parasita , Humanos , América Latina/epidemiologia , Doenças Negligenciadas/epidemiologia , Doenças Negligenciadas/imunologia , Medicina TropicalRESUMO
Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs. BIOLOGICAL SIGNIFICANCE: Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.
Assuntos
Larva/metabolismo , Estágios do Ciclo de Vida , Proteômica/métodos , Animais , Cestoides/química , Infecções por Cestoides/diagnóstico , Infecções por Cestoides/tratamento farmacológico , Proteínas de Helminto/análise , Proteínas de Helminto/fisiologia , Humanos , Mesocestoides/química , Reprodução Assexuada , Diferenciação SexualRESUMO
BACKGROUND: The neglected disease cystic echinococcosis is caused by larval Echinococcus granulosus flatworms, which form bladder-like hydatid cysts in liver, lungs, and other organs. SOURCES OF DATA: Published literature. AREAS OF AGREEMENT: Establishing larvae are susceptible to antibody-dependent killing, as attested by successful animal vaccination, whereas once established they are partially protected by the so-called laminated layer. Host responses are Th2 dominated, with a Th1 component. Diagnostic antigens from cyst fluid are known, but responses appear absent in one-fifth of patients. AREAS OF CONTROVERSY: Is evasion mainly based on induction of Th2 or regulatory responses by the parasite? GROWING POINTS: The parasite induces regulatory responses. The laminated layer has immune-regulatory properties. AREAS TIMELY FOR DEVELOPING RESEARCH: Develop tools for functional genomics; characterize immunologically interesting proteins suggested by genomic information; analyse infection in broader context of granulomatous responses; identify molecules secreted/excreted by intact larvae/cysts towards their outside, including diffusible immune-regulators.
Assuntos
Antígenos de Helmintos/imunologia , Equinococose/imunologia , Equinococose/parasitologia , Echinococcus granulosus/imunologia , Interações Hospedeiro-Parasita/imunologia , Animais , Antígenos de Helmintos/fisiologia , Proteínas de Helminto/fisiologia , Humanos , Imunidade Celular , LarvaRESUMO
The hydatid fluid (HF) that fills Echinococcus multilocularis metacestode vesicles is a complex mixture of proteins from both parasite and host origin. Here, a LC-MS/MS approach was used to compare the HF composition of E. multilocularis H95 and G8065 isolates (EmH95 and EmG8065, respectively), which present differences in terms of growth and fertility. Overall, 446 unique proteins were identified, 392 of which (88%) were from parasite origin and 54 from culture medium. At least 256 of parasite proteins were sample exclusive, and 82 of the 136 shared proteins presented differential abundance between E. multilocularis isolates. The parasite's protein repertoires in EmH95 and EmG8065 HF samples presented qualitative and quantitative differences involving antigens, signaling proteins, proteolytic enzymes, protease inhibitors and chaperones, highlighting intraspecific singularities that could be correlated to biological features of each isolate. The repertoire of medium proteins found in the HF was also differential between isolates, and the relevance of the HF exogenous protein content for the parasite's biology is discussed. The repertoires of identified proteins also provided potential molecular markers for important biological features, such as parasite growth rate and fertility, as well potential protein targets for the development of novel diagnostic and treatment strategies for alveolar echinococcosis. BIOLOGICAL SIGNIFICANCE: E. multilocularis metacestode infection of mammal hosts involve complex interactions mediated by excretory/secretory (ES) products. The hydatid fluid (HF) that fills the E. multilocularis metacestode vesicles contains complex repertoires of parasite ES products and host proteins that mediate important molecular interactions determinant for parasite survival and development, and, consequently, to the infection outcome. HF has been also extensively reported as the main source of proteins for the immunodiagnosis of echinococcosis. The performed proteomic analysis provided a comprehensive profiling of the HF protein composition of two E. multilocularis isolates. This allowed us to identify proteins of both parasite and exogenous (medium) origin, many of which present significant differential abundances between parasite isolates and may correlate to their differential biological features, including fertility and growth rate.
Assuntos
Echinococcus multilocularis/química , Proteínas de Helminto/análise , Proteômica/métodos , Animais , Biomarcadores/análise , Líquidos Corporais/química , Equinococose/diagnóstico , Equinococose/imunologia , Fertilidade , Crescimento , Proteínas de Helminto/fisiologia , Interações Hospedeiro-Parasita , Especificidade da EspécieRESUMO
Helminths cause a number of medical and agricultural problems and are a major cause of parasitic infections in humans, animals and plants. Comparative analysis of helminth genes and genomes are important to understand the genomic biodiversity and evolution of parasites and their hosts in terms of different selective pressures in their habitats. The interactions between the infective organisms and their hosts are mediated in large part by secreted proteins, known collectively as the "secretome". Proteins secreted by parasites are able to modify a host's environment and modulate their immune system. The composition and function of this set of proteins varies depending on the ecology, lifestyle and environment of an organism. The present study aimed to predict, in silico, the secretome in 44 helminth species including Nematoda (31 species) and Platyhelminthes (13 species) and, understand the diversity and evolution of secretomes. Secretomes from plant helminths range from 7.6% (943 proteins) to 13.9% (2,077 proteins) of the filtered proteome with an average of 10.2% (1,412 proteins) and from free-living helminths range from 4.4% (870 proteins) to 13% (3,121 proteins) with an average of 9.8% (2,126 proteins), respectively, and thus are considerably larger secretomes in relation to animal helminth secretomes which range from 4.2% (431 proteins) to 11.8% (2,419 proteins) of the proteomes, with an average of 7.1% (804 proteins). Across 44 secretomes in different helminth species, we found five conserved domains: (i) PF00014 (Kunitz/Bovine pancreatic trypsin inhibitor domain), (ii) PF00046 (Homeobox domain), (iii) PF00188 (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), (iv) PF00085 (Thioredoxin) and (v) PF07679 (Immunoglobulin I-set domain). Our results detected secreted proteins associated with invasion, infection, adhesion and immunoregulation processes as protease inhibitors and cytokines, among other functions. In summary, this study will contribute towards the understanding of host-parasite interactions and possibly identify new molecular targets for the treatment or diagnosis of helminthiases.
Assuntos
Proteínas de Helminto/metabolismo , Helmintos/metabolismo , Animais , Biodiversidade , Sequência Conservada , Genoma , Proteínas de Helminto/química , Proteínas de Helminto/fisiologia , Helmintos/classificação , Helmintos/genética , Helmintos/fisiologia , Interações Hospedeiro-Parasita , Estilo de Vida , Filogenia , Plantas/parasitologia , Domínios Proteicos , Sinais Direcionadores de Proteínas/fisiologia , Especificidade da EspécieRESUMO
Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.
Assuntos
Cisticercose/imunologia , Cysticercus/imunologia , Proteínas de Helminto/imunologia , Imunoglobulina G/biossíntese , Doenças dos Suínos/parasitologia , Taenia solium/imunologia , Animais , Antígenos de Helmintos/imunologia , Catepsina L/imunologia , Cisticercose/parasitologia , Cysticercus/fisiologia , Proteínas de Helminto/fisiologia , Imunoglobulina G/efeitos dos fármacos , Peso Molecular , Suínos , Doenças dos Suínos/imunologia , Taenia solium/fisiologia , Vacinas/imunologia , Vacinas/farmacologiaRESUMO
Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-beta production and the expansion of CD4(+)CD25(+)Foxp3(+) cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.
Assuntos
Células Dendríticas/imunologia , Fasciola hepatica/imunologia , Proteínas de Helminto/fisiologia , Tolerância Imunológica , Células Mieloides/imunologia , Animais , Citocinas/biossíntese , Feminino , Fatores de Transcrição Forkhead/análise , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Células Th2/imunologia , Receptores Toll-Like/fisiologiaRESUMO
FK506 (tacrolimus) and polyketide macrolides such as rapamycin and its derivates bind to FK506-binding proteins (FKBPs). These proteins display a peptidyl-prolyl rotamase function that is believed to catalyze protein folding and they are well-validated anti-proliferative drug targets in certain pathogenic microorganisms, and their functions have been characterized in parasitic protozoa. However, much less is known in helminths and trials with rapalogs on cestoda have not yet been reported. Due to a growing need for new treatment options for human cystic echinococcosis, the in vitro efficacy of rapalogs in Echinococcus granulosus was investigated. We determined the effect of ramapycin, FK506 and everolimus against this cestode, demonstrating their protoscolicidal ability. Also, we observed synergic scolicidal actions during combined therapy with rapalogs plus cyclosporine A, proposing dual administration of drugs to improve pharmacological effects in vivo. We have identified an E. granulosus (Eg)-fkb1 gene that encodes Eg-FKBP, an archetypal protein of the FKBP family, which includes all residues implicated in the binding of pharmacological ligands, in the enzymatic activity and in interactions with possible target proteins. Levels of Eg-fkb1 mRNA are over-expressed by acid but not rapalog treatment. We also described the presence of receptor-operated calcium channels in the larval stage, suggesting that exogenous ligands may dissociate the interaction of Eg-FKBP from these intracellular channels, enhancing the activity of the Ca(2+) release and interfering with their normal regulatory functions. As rapamycin sensitivity is the major criterion used to detect targets of rapamycin kinase, we identified and analyzed in silico critical residues of putative homologs in the Echinococcus genome. These preliminary results will allow us to continue subsequent studies that could reveal the precise intracellular functions of Eg-FKBP, providing greater knowledge for further identification of downstream target proteins, a promising target for chemotherapy of cystic echinococcosis.
Assuntos
Anti-Helmínticos/farmacologia , Cálcio/metabolismo , Echinococcus granulosus/efeitos dos fármacos , Echinococcus granulosus/fisiologia , Proteínas de Helminto/fisiologia , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Ciclosporina/farmacologia , DNA de Helmintos/química , DNA de Helmintos/genética , Sinergismo Farmacológico , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Everolimo , Perfilação da Expressão Gênica , Proteínas de Helminto/genética , Homeostase , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Sirolimo/análogos & derivados , Análise de Sobrevida , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/genética , Estados UnidosRESUMO
As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 degrees C, in the range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.
Assuntos
Echinococcus granulosus/enzimologia , Lectinas/química , N-Acetilgalactosaminiltransferases/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Células COS/química , Células COS/metabolismo , Bovinos , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/parasitologia , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cobre/fisiologia , DNA Complementar/genética , DNA de Helmintos/genética , Equinococose/enzimologia , Equinococose/veterinária , Proteínas de Helminto/biossíntese , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Concentração de Íons de Hidrogênio , Lectinas/genética , Manganês/metabolismo , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/biossíntese , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/fisiologia , Peptídeos/genética , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Especificidade por Substrato/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The complete sequence of SmCys, a cystatin expressed by Schistosoma mansoni, was obtained. Constructs of SmCys consisting of deletions of 10 and 20 amino acid residues from the N-terminal of the full length recombinant protein, were cloned in the pQE-30 vector, expressed in Escherichia coli and assayed for inhibitory activity against papain. Kinetic analysis showed that SmCys -10 and SmCys -20 had K(i) values of 0.7391 and 4.9154, respectively, as compared to 0.0647, displayed by the full length recombinant. Protease inhibition by SmCys was also observed in vivo. When the recombinant products were incubated during 7 days with live schistosomula in the presence of red blood cells, only the full length product could completely inhibit the formation of haemozoin, a dark pigment formed as a by-product of haemoglobin digestion. The sequence data of the recombinant SmCys proteins were used for the construction of molecular models, which were then subjected to molecular dynamics for 2ns. In comparison to the full length, the models corresponding to the truncated constructs, showed a distinctive change on the surface charge distribution. This parameter was more pronounced in SmCys -20, which also displayed a significant displacement of the inhibitory domain, a result which could explain the kinetic data in terms of the loss of attachment sites. These changes correlated well with the progressive lack of inhibition observed for the recombinant deletion constructs, in vitro and in vivo.
Assuntos
Cistatinas/química , Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/química , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Cistatinas/genética , Cistatinas/isolamento & purificação , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/isolamento & purificação , Inibidores de Cisteína Proteinase/farmacologia , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/fisiologia , Hemeproteínas/metabolismo , Hemoglobinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Papaína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Schistosoma mansoni/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.
Assuntos
Proteínas de Ligação ao Cálcio , Membrana Eritrocítica/metabolismo , Proteínas de Helminto/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas Periplásmicas de Ligação , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Endotélio Vascular/parasitologia , Membrana Eritrocítica/parasitologia , Imunofluorescência , Galactose/metabolismo , Coração/parasitologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/fisiologia , Humanos , Soros Imunes/imunologia , Imuno-Histoquímica , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Coelhos , Trypanosoma cruzi/químicaRESUMO
Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells.
Assuntos
Proteínas de Transporte/análise , Proteínas de Helminto/análise , Proteínas de Membrana Transportadoras , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos/imunologia , DNA Complementar , Proteínas de Transporte de Ácido Graxo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Estágios do Ciclo de Vida , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/imunologiaRESUMO
Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system
Assuntos
Animais , Feminino , Camundongos , Schistosoma mansoni/imunologia , Proteínas Recombinantes , Anticorpos Anti-Helmínticos/fisiologia , Proteínas de Helminto/fisiologia , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas de Transporte , Proteínas de Helminto/isolamento & purificação , Western Blotting , Sequência de Aminoácidos , Vacinação , DNA Complementar , Modelos Animais , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Ácidos GraxosRESUMO
We and others have previously shown that nematodes or nematode products can stimulate or inhibit the generation of lymphocyte responses, suggesting that nematodes exert diverse effects on the developing immune responses of their host. In this study we examined the immunomodulatory effect of a soluble extract of Nippostrongylus brasiliensis (adult worm homogenate [AWH]) on B-cell responsiveness. We found that the extract inhibited the proliferation of B cells to lipopolysaccharide (LPS) stimulation in a dose-dependent manner. This effect was specific to B cells, since the extract did not inhibit T-cell proliferation to concanavalin A or anti-CD3 stimulation. The data presented here confirm that the extract is not toxic to B cells. We present evidence that the active factor is proteinaceous in nature and that the inhibitory activity is restricted to the adult stage of Nb. The extract does not appear to interfere with early activation events since it can be added up to 48 h after LPS stimulation, and it inhibited responses to phorbol myristate acetate and ionomycin. Furthermore, the proliferation of B cells to other activators was also inhibited by AWH. This observation shows that the inhibitory activity of AWH is not restricted to LPS-mediated B-cell proliferation. We present evidence that, in the absence of accessory cells, the inhibitory effect of the extract was ablated. This observation shows that the activity of AWH is not mediated directly on B cells but is mediated via the production of negative signals from accessory cells (macrophages), which affect a downstream pathway required by all B-cell activators tested. These effects on B-cell and accessory cell function are likely to have a significant effect on the outcome of infections experienced concurrently.
Assuntos
Linfócitos B/imunologia , Proteínas de Helminto/fisiologia , Ativação Linfocitária , Nippostrongylus/fisiologia , Animais , Feminino , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase C/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.
Assuntos
Células 3T3/enzimologia , Actinas/fisiologia , Cloranfenicol O-Acetiltransferase/metabolismo , Echinococcus/genética , Proteínas de Helminto/fisiologia , Regiões Promotoras Genéticas/fisiologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Genes Reporter , Proteínas de Helminto/química , Proteínas de Helminto/genética , Camundongos , Regiões Promotoras Genéticas/genética , Relação Estrutura-AtividadeRESUMO
The 50-30 kDa fraction isolated from the excretory/secretory products (E/S) of the nematode Nippostrongylus brasiliensis significantly decreased the amplitude of contraction of segments of uninfected rat intestine when injected into the lumen of the segments maintained in an organ bath. Dot blot analysis of the fraction suggested that it was similar in immunoreactivity to porcine vasoactive intestinal polypeptide (VIP). When antiserum to porcine VIP was mixed with N. brasiliensis E/S and the mixtures were injected into the lumen of segments of rat intestine, the inhibitory effect of the E/S on amplitude of contraction decreased. When physiological concentrations of porcine VIP (12.9 pmol/ml) were injected into the lumen of segments of uninfected rat intestine the amplitude of contraction decreased significantly. Western blot analysis of the E/S, using antiserum to porcine VIP, recognized a 30 kDa protein in the E/S and also in whole worm homogenate suggesting that synthesis of the peptide occurs inside the nematode. Peptide histidine isoleucine (PHI)-like immunoreactivity was detected in a 68 kDa fraction of the E/S and the homogenate but this fraction did not affect the amplitude of contractions of the intestine.