RESUMEN
A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.
Asunto(s)
Proteínas del Helminto/metabolismo , Larva/enzimología , Serina Endopeptidasas/metabolismo , Strongyloides ratti/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Medios de Cultivo/química , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Sueros Inmunes/química , Intestinos/parasitología , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Prolil Oligopeptidasas , Inhibidores de Proteasas/farmacología , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteómica , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de Proteína , Serina Endopeptidasas/genética , Estadísticas no Paramétricas , Strongyloides ratti/genética , Strongyloides ratti/crecimiento & desarrollo , Estrongiloidiasis/parasitologíaRESUMEN
Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.
Asunto(s)
Genómica , Strongyloides/genética , Estrongiloidiasis/genética , Simbiosis/genética , Animales , Evolución Biológica , Humanos , Estadios del Ciclo de Vida/genética , Strongyloides/patogenicidad , Estrongiloidiasis/parasitología , Transcriptoma/genéticaRESUMEN
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.
Asunto(s)
Proteínas del Helminto/inmunología , Interacciones Huésped-Parásitos , Factores Inhibidores de la Migración de Macrófagos/inmunología , Strongyloides ratti/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/aislamiento & purificación , Movimiento Celular , Reacciones Cruzadas , Escherichia coli/química , Escherichia coli/genética , Femenino , Citometría de Flujo , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Humanos , Inmunoglobulina G/sangre , Interleucina-10/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/aislamiento & purificación , Macrófagos/inmunología , Masculino , Datos de Secuencia Molecular , Monocitos/inmunología , Filogenia , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Strongyloides ratti/genética , Strongyloides ratti/patogenicidad , Estrongiloidiasis/inmunología , Estrongiloidiasis/parasitología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Strongyloidiasis is a neglected disease that is prevalent mainly in tropical and subtropical regions. It is caused by intestinal nematodes of the genus Strongyloides. Due to the rise in worldwide travel, infections are increasingly encountered in non-endemic regions. Diagnosis is hampered by insensitive and laborious detection methods. A universal Strongyloides species real-time PCR was developed with an internal competitive control system. The 95% limit of detection as determined by probit analysis was one larva per PCR equivalent to 100 larvae per 200 mg stool. The assay proved to be 100% specific as assessed using a panel of parasites and bacteria and thus might be useful in the diagnostic setting as well as for Strongyloides research.
Asunto(s)
Parasitología/métodos , Reacción en Cadena de la Polimerasa/métodos , Strongyloides/genética , Strongyloides/aislamiento & purificación , Estrongiloidiasis/diagnóstico , Animales , Humanos , Parasitología/normas , Reacción en Cadena de la Polimerasa/normas , Ratas , Ratas Wistar , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/metabolismo , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Mucosa Intestinal/metabolismo , Strongyloides ratti/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Femenino , Proteínas de Choque Térmico Pequeñas/química , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/aislamiento & purificación , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Humanos , Mucosa Intestinal/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Datos de Secuencia Molecular , Monocitos/inmunología , Monocitos/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Strongyloides ratti/crecimiento & desarrollo , Strongyloides ratti/inmunologíaRESUMEN
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.