RESUMO
Angiostrongylus cantonensis is a parasitic nematode and the main causative agent of human cerebral eosinophilic meningoencephalitis (EoM). A definitive diagnosis of EoM usually requires serologic or molecular analysis of the patient's clinical sample. Currently, a 31â¯kDa antigen is used in immunological tests for this purpose, however as a crude antigen preparation it may present cross-reactivity with other helminthic infections, especially echinococcosis. Heterologous expression studies using prokaryotic systems failed on producing antigenic proteins. The aim of this study was to express and purify three recombinant glycoproteins representing A. cantonensis antigens: ES-7, Lec-5, and 14-3-3, in Chinese hamster ovary (CHO) cells and ES-7 in human embryonic kidney (HEK) cells to develop a source of specific antigens to be used in the diagnosis of angiostrongyliasis. The potential diagnostic value of these three proteins was subsequently characterized in one- and two-dimensional electrophoresis and Western blot to dot blot analyses, with Angiostrongylus-positive sera, normal human sera (NHS), and a pool of Echinococcus-positive sera (included as a specificity control) used for detection. In addition, recognition of these three proteins following treatment with N-glycosidase F was examined. The ES-7 proteins that were expressed in HEK and CHO cells, and the Lec-5 protein that was expressed in CHO cells, were specifically recognized by A. cantonensis-positive sera in the 2D electrophoresis analysis. This recognition was shown to be dependent on the presence of glycidic portions, making mammalian cells a very promising source of heterologous expression antigenic proteins from Angiostrongylus.
Assuntos
Angiostrongylus cantonensis/genética , Antígenos de Helmintos/biossíntese , Proteínas de Helminto/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Antígenos de Helmintos/genética , Western Blotting , Células CHO , Clonagem Molecular , Cricetulus , Expressão Gênica , Células HEK293 , Proteínas de Helminto/genética , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Testes Sorológicos/métodos , Infecções por Strongylida/diagnósticoRESUMO
GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.
Assuntos
Anticorpos Monoclonais/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Glutationa Transferase/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Schistosoma japonicum/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Avidina/química , Biotina/química , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Hibridomas/imunologia , Hibridomas/patologia , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/enzimologia , Baço/citologia , Baço/imunologiaRESUMO
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
Assuntos
Expressão Gênica , Vetores Genéticos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas , Animais , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/genética , Fusão Gênica Artificial , Clonagem Molecular , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Mutagênese , Micobacteriófagos/genética , Mycobacterium smegmatis/genética , Plasmídeos , Reação em Cadeia da Polimerase , Schistosoma mansoni/genética , Análise de Sequência de DNARESUMO
The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface.
Assuntos
Alérgenos/biossíntese , Antígenos de Helmintos/biossíntese , Expressão Gênica , Proteínas de Helminto/biossíntese , Schistosoma mansoni/crescimento & desenvolvimento , Estruturas Animais/química , Animais , DNA de Helmintos/química , DNA de Helmintos/genética , Perfilação da Expressão Gênica , Hibridização In Situ , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Schistosoma mansoni/química , Schistosoma mansoni/genética , Análise de Sequência de DNARESUMO
Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.
Assuntos
Perfilação da Expressão Gênica , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Animais , Antígenos de Helmintos/biossíntese , DNA de Helmintos/química , DNA de Helmintos/genética , Etiquetas de Sequências Expressas , Proteínas de Helminto/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquistossomose mansoni/prevenção & controle , Análise de Sequência de DNA , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Helminths secrete several molecules that can modulate the immune responses, favoring their evasion and perpetuate their survival in the host. These molecules interfere with antigen presentation, cell proliferation and activation, antibody production, cause cell death, and stimulate regulatory responses. Here, we focus on some helminth products and address their immunomodulatory effects in the host immune system and, also, we describe some anti-inflammatory properties of an Ascaris suum-derived immunomodulatory molecule, named PAS-1. This protein is a 200-kDa molecule isolated by affinity chromatography using MAIP-1 (monoclonal antibody which recognizes PAS-1), coupled to Sepharose 4B. It suppresses the inflammatory responses in murine models of delayed-type hypersensitivity, lung allergic inflammation and LPS-induced inflammation into air pouches. PAS-1 also stimulates the secretion of regulatory cytokines such as IL-10 and TGF-beta and primes IFN-gamma-secreting CD8+ and IL-10/ TGF-beta-secreting CD4+CD25+ cell clones that avoid the lung inflammation. Thus, this protein is a potent immunomodulatory component that may be used for therapeutic interventions in inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Helminto/farmacologia , Helmintos/metabolismo , Imunidade/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Inflamação/tratamento farmacológico , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Ascaris suum/metabolismo , Citocinas/biossíntese , Citocinas/farmacologia , Citocinas/uso terapêutico , Proteínas de Helminto/biossíntese , Proteínas de Helminto/uso terapêutico , Fatores Imunológicos/biossíntese , Fatores Imunológicos/uso terapêutico , Inflamação/imunologia , Linfócitos T/imunologiaRESUMO
Many parasites undergo sudden changes in environmental conditions at some stage during their life cycle. The molecular response to this variation is characterised by a rapid transcriptional activation of a specific set of genes coding for proteins generically known as stress proteins. They appear to be also involved in various biological processes including cell proliferation and differentiation. The platyhelminth parasite, Mesocestoides corti (Cestoda) presents important properties as a model organism. Under stress conditions, key molecules involved in metabolic pathways as well as in the growth and differentiation of the parasite can be identified. 2D protein expression profile of tetrathyridia of M. corti, submitted to nutritional starvation and cold stress is described, as well as the recovery pattern. A set of specifically expressed proteins was observed in each experimental condition. Quantitative and qualitative differences and stress recovery pattern are also reported. This work makes evident the high plasticity and resistance to extreme environmental conditions of these parasites at the molecular level.
Assuntos
Temperatura Baixa , Proteínas de Choque Térmico/análise , Proteínas de Helminto/análise , Mesocestoides/metabolismo , Animais , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/química , Proteínas de Helminto/genética , Ponto Isoelétrico , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Mesocestoides/genética , Mesocestoides/crescimento & desenvolvimento , Camundongos , Peso Molecular , Proteômica , Coloração pela PrataRESUMO
In higher eukaryotes, histone gene expression is coupled to DNA replication during the S-phase of the cell cycle. This coupling is primarily controlled at the transcriptional level. Considering the basal phylogenic position of platyhelminthes in the bilateria phylum, we have cloned a partial sequence of the histone H4 gene of Mesocestoides corti and studied its expression during the post larval development of this endoparasitic platyhelminth. In in vitro trypsin-induced tetrathyridia development to segmented adult worm, we found that histone H4 is expressed concomitantly with DNA synthesis throughout all stages of development. DNA synthesis and histone H4 mRNA levels were sharply increased at 24 h after inducing development. Afterwards, tetrathyridia grew in length from days 4 to 12 of development as proliferative cells gradually increased in number. Consequently, during this period of development histone H4 mRNA levels were upregulated. Taken together these results suggest that a replication-dependent expression pattern of histone H4 occurs in ancient bilateria, such as platyhelminthes, as previously observed in higher eukaryotes.
Assuntos
Replicação do DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/biossíntese , Histonas/genética , Mesocestoides/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Masculino , Mesocestoides/embriologia , Mesocestoides/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
We report the oral vaccination of SWISS mice with an Aro attenuated Salmonella enterica var. Typhimurium vaccine strain expressing the 14-kDa Schistosoma mansoni antigen, Sm14. Bacterial adjuvants, including (i) Lactococcus lactis expressing interleukin-12 (IL-12) and (ii) Lactobacillus delbrueckii UFV-H2b20, were also employed in oral immunization assays. Detection assays to specific IgG and IgA anti-Sm14 antibodies were performed to evaluate humoral immune responses in vaccinated mice. An increase in specific IgG titers was observed; however, no IgA production was detected. The protection levels against schistosomiasis (34.9-49.5%) obtained with all experimental formulations in this work were very similar to values reported by previous studies, which used purified recombinant Sm14 for parenteral vaccination of mice. There was a slight reduction in hepatic granulomas of mice vaccinated with Salmonella. Oogram studies showed diminished numbers of S. mansoni eggs in the intestinal wall of vaccinated mice, but individual female worm fecundity did not seem to be affected by our immunization protocol.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras/imunologia , Vacinas contra Salmonella/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose/imunologia , Animais , Antígenos de Helmintos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Proteínas de Transporte de Ácido Graxo , Feminino , Proteínas de Helminto/biossíntese , Proteínas de Helminto/efeitos dos fármacos , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Camundongos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Esquistossomose/prevenção & controleRESUMO
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-AcetilgalactosaminiltransferaseAssuntos
Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Proteínas de Helminto/biossíntese , Tropomiosina/biossíntese , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Echinococcus/genética , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Larva/genética , Larva/crescimento & desenvolvimento , Tropomiosina/genéticaRESUMO
Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared. DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed. The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20. Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns. According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years. When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity. Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.
Assuntos
Antígenos de Helmintos/genética , Echinococcus/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/biossíntese , Sequência de Bases , Clonagem Molecular , Echinococcus/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Genes de Helmintos , Vetores Genéticos , Proteínas de Helminto/biossíntese , Helmintíase/sangue , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
The homeobox-containing gene EgHbx3 from Echinococcus granulosus is expressed in the stalk of protoscoleces. The expression of the homeobox-containing gene EgHbx3 from E. granulosus was studied in protoscoleces by in situ hybridisation techniques. A method for performing in situ hybridisations on whole mounted hydatic material is described. Our results show a clear positive signal at the protoscolex stalk, when using the antisense EgHbx3 probe. This signal is particularly strong in the proximal end of the stalk (with respect to the protoscolex).
Assuntos
Echinococcus/genética , Genes de Helmintos , Genes Homeobox , Proteínas de Helminto/biossíntese , Animais , Echinococcus/anatomia & histologia , Expressão Gênica , Hibridização In Situ , Dados de Sequência MolecularRESUMO
The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and the parasite's ability to survive within the definitive host. A focus will be the recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.