RESUMO
Paramyosin is a muscular protein exclusively found in invertebrate species, which has been proposed as a vaccine candidate against infections with Schistosoma mansoni and Brugia malayi. Here, we report the studies on the distribution of Onchocerca volvulus paramyosin, designated OvPmy, in different O. volvulus stages by immunohistochemistry using rabbit antibodies raised against the recombinant OvPmy protein as well as the induction of the human humoral immune response to OvPmy. To conduct the studies, OvPmy was expressed in Escherichia coli as a fusion protein to raise the rabbit antibodies. The recombinant OvPmy was tested in immunoblots using sera from individuals living in an area hyperendemic for onchocerciasis in Liberia, West Africa. The antibodies used here localised paramyosin exclusively in the muscle tissue of O. volvulus as well as Onchocerca ochengi. No extracellular compartments, such as the cuticle or the lumina of the pseudocoeloma cavity, were labelled; however, labelling was seen in microfilarial fragments taken up by host immune cells, such as giant cells. It was recognised by anti-paramyosin antibodies of a group of onchocerciasis patients.
Assuntos
Onchocerca volvulus/química , Oncocercose/imunologia , Tropomiosina/análise , Animais , Anticorpos Anti-Helmínticos/sangue , Doenças Endêmicas , Humanos , Imuno-Histoquímica/métodos , Libéria/epidemiologia , Dados de Sequência Molecular , Músculos/química , Oncocercose/epidemiologia , CoelhosRESUMO
In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model.
Assuntos
Clonagem Molecular , DNA de Helmintos/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Imunização , Onchocerca volvulus/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Sequência de Bases , Bovinos , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Imuno-Histoquímica , Íntrons , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Tempo , Vacinas Sintéticas/imunologiaRESUMO
The secreted protein acidic and rich in cysteine (SPARC), found in the excretory/secretory products of Strongyloides ratti, is most strongly expressed in parasitic females. Since SPARC proteins are involved in the modulation of cell-matrix interactions, a role of the secreted S. ratti SPARC (Sr-SPARC) in the manifestation of the parasite in the host's intestine is postulated. The full-length cDNA of Sr-SPARC was identified and the protein was recombinantly expressed. The purified protein was biologically active, able to bind calcium, and to attach to mucosa-associated human cells. Addition of Sr-SPARC to an in vitro mucosal three-dimensional-cell culture model led to a time-dependent release of the cytokines TNF-α, IL-22, IL-10 and TSLP. Of importance, exposure with Sr-SPARC fostered wound closure in an intestinal epithelial cell model. Here, we demonstrate for the first time that SPARC released from the nematode is a multifunctional protein affecting the mucosal immune system.
Assuntos
Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Mucosa Intestinal/imunologia , Osteonectina/metabolismo , Strongyloides ratti/fisiologia , Estrongiloidíase/imunologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Proteínas de Helminto/química , Humanos , Mucosa Intestinal/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Osteonectina/química , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Estrongiloidíase/parasitologia , CicatrizaçãoRESUMO
The cellular redox state is important for the regulation of multiple functions and is essential for the maintenance of cellular homeostasis and antioxidant defense. In the excretory/secretory (E/S) products of Strongyloides ratti and Trichuris suis sequences for thioredoxin (Trx) and Trx-like protein (Trx-lp) were identified. To characterize the antioxidant Trx-lp and its interaction with the parasite's mucosal habitat, S. ratti and T. suis Trx-lps were cloned and recombinantly expressed. The primary antioxidative activity was assured by reduction of insulin and IgM. Further analysis applying an in vitro mucosal 3D-cell culture model revealed that the secreted Trx-lps were able to bind to monocytic and intestinal epithelial cells and induce the time-dependent release of cytokines such as TNF-α, IL-22, and TSLP. In addition, the redox proteins also possessed chemotactic activity for monocytic THP-1 cells and fostered epithelial wound healing activity. These results confirm that the parasite-secreted Trx-lps are multifunctional proteins that can affect the host intestinal mucosa.
RESUMO
River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.
Assuntos
Filariose/prevenção & controle , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Onchocerca volvulus/enzimologia , Onchocerca volvulus/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/genética , Camundongos Endogâmicos BALB C , Onchocerca volvulus/genética , Carga Parasitária , Plasmídeos/administração & dosagem , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Strongyloidiasis is a tropical parasitosis characterized by an alternation between free-living and parasitic stages, and by long-term infection via autoinfection. Since invasion and evasion processes of helminth parasites are substantially attained by the involvement of excretory-secretory products, we identified and characterized the 13.5 kDa macrophage migration inhibitory factor (MIF)-like protein in Strongyloides ratti. Sra-MIF is mainly secreted from the infective stage larvae (iL3), while the transcript was found at lower levels in parasitic and free-living females. Sequence analysis of the full-length cDNA showed the highest homology to the human pathogen Strongyloides stercoralis, and both are related to the MIF type-2. Unlike other mif genes, the Sra-mif includes no intron. The protein was recombinantly expressed in Escherichia coli and purified. Sra-MIF exhibited no in vitro tautomerase activity. The exposure of Sra-MIF to the host immune system is confirmed by high IgG reactivities found in the hosts' sera following infection or immunization. Flow cytometric analysis indicated the binding of Sra-MIF to the monocytes/macrophage lineage but not to peripheral lymphocytes. After exposure to Sra-MIF, monocytes released IL-10 but not TNF-alpha suggesting the involvement of the secreted parasite MIF in host immune responses.
Assuntos
Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Fatores Inibidores da Migração de Macrófagos/imunologia , Strongyloides ratti/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/isolamento & purificação , Movimento Celular , Reações Cruzadas , Escherichia coli/química , Escherichia coli/genética , Feminino , Citometria de Fluxo , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Humanos , Imunoglobulina G/sangue , Interleucina-10/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Macrófagos/imunologia , Masculino , Dados de Sequência Molecular , Monócitos/imunologia , Filogenia , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Strongyloides ratti/genética , Strongyloides ratti/patogenicidade , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
In a search for molecules involved in the interaction between intestinal nematodes and mammalian mucosal host cells, we performed MS to identify excretory-secretory proteins from Strongyloides ratti. In the excretory-secretory proteins of the parasitic female stage, we detected, in addition to other peptides, peptides homologous with the Caenorhabditis elegans heat shock protein (HSP)-17, named Sra-HSP-17.1 (â¼ 19 kDa) and Sra-HSP-17.2 (â¼ 18 kDa), with 49% amino acid identity. The full-length cDNAs (483 bp and 474 bp, respectively) were identified, and the genomic organization was analyzed. To allow further characterization, the proteins were recombinantly expressed and purified. Profiling of transcription by quantitative real-time-PCR and of protein by ELISA in various developmental stages revealed parasitic female-specific expression. Sequence analyses of both the DNA and amino acid sequences showed that the two proteins share a conserved α-crystallin domain and variable N-terminals. The Sra-HSP-17s showed the highest homology with the deduced small HSP sequence of the human pathogen Strongyloides stercoralis. We observed strong immunogenicity of both proteins, leading to strong IgG responses following infection of rats. Flow cytometric analysis indicated the binding of Sra-HSP-17s to the monocyte-macrophage lineage but not to peripheral lymphocytes or neutrophils. A rat intestinal epithelial cell line showed dose-dependent binding to Sra-HSP-17.1, but not to Sra-HSP-17.2. Exposed monocytes released interleukin-10 but not tumor necrosis factor-α in response to Sra-HSP-17s, suggesting the possible involvement of secreted female proteins in host immune responses.
Assuntos
Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Mucosa Intestinal/metabolismo , Strongyloides ratti/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Feminino , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/isolamento & purificação , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Humanos , Mucosa Intestinal/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Dados de Sequência Molecular , Monócitos/imunologia , Monócitos/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Strongyloides ratti/crescimento & desenvolvimento , Strongyloides ratti/imunologiaRESUMO
Strongyloides stercoralis and S. ratti are intestinal parasitic nematodes infecting rats and humans, respectively. Both present extraordinary life cycles comprising a free-living generation in addition to parasitic stages. In search of molecules possibly involved in parasite-host interaction, we performed mass spectrometry to identify excretory/secretory products of S. ratti. Amongst others we detected homologs of the heat shock proteins HSP10 and HSP60 (Sr-HSP10 and Sr-HSP60). HSPs are well known as chaperones involved in stress responses of cells, but recent studies suggest additional roles of small HSPs for parasite biology including immune modulation. To characterise Sr-HSP10, we cloned its full-length cDNA, analysed the genomic organisation, tested its presumptive role as an interaction partner of Sr-HSP60, studied its transcription in the parasite, and expressed the protein to test its immune responses. The cDNA contains an open reading frame of 330bp encoding a polypeptide of 110 amino acids with an approximate molecular weight of 10kDa. The Sr-HSP10 protein is highly homologous to that of the human pathogen S. stercoralis with only eight amino acid substitutions. Analysis of the genomic organisation of the Sr-HSP10 locus revealed that the gene is linked head-to-head to the gene encoding Sr-HSP60, and both share a bidirectional promoter. RT-PCR experiments indicated potential independent expression of the Sr-HSPs genes. In situ hybridisation results demonstrate Sr-HSP10 transcription in the gut area. Mammalian and yeast two-hybrid assays show dimerisation of Sr-HSP10, but no binding to recombinant Sr-HSP60. Immunisation experiments finally revealed a strong immunogenicity of Sr-HSP10 and provided evidence for a role in regulating the host-parasite interaction.