RESUMO
Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yieldingâ¼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.
Assuntos
Ancylostomatoidea/crescimento & desenvolvimento , Trato Gastrointestinal/parasitologia , Proteínas de Helminto/metabolismo , Estágios do Ciclo de Vida , Proteoma/análise , Ancylostomatoidea/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNARESUMO
The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.
Assuntos
Duodeno/parasitologia , Lectinas Tipo C/metabolismo , Nematospiroides dubius/crescimento & desenvolvimento , Nippostrongylus/crescimento & desenvolvimento , Animais , Biblioteca Gênica , Interações Hospedeiro-Parasita , Larva/metabolismo , Lectinas Tipo C/genética , Estágios do Ciclo de Vida , Camundongos , Dados de Sequência Molecular , Nematospiroides dubius/genética , Nematospiroides dubius/metabolismo , Nippostrongylus/genética , Nippostrongylus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Helminth infections at mucosal and tissue sites strongly polarize towards Th2 immune responses, following pathways which have yet to be elucidated. We investigated whether dendritic cells (DC) exposed to gastrointestinal nematodes induce Th2 differentiation and, if so, whether this outcome reflects the absence of DC activation (the default hypothesis). We studied secreted proteins from the parasite Nippostrongylus brasiliensis, which induce Th2 development in vivo without live infection. Murine bone marrow-derived DC pulsed with N. brasiliensis excretory/secretory antigen (NES) can, on transfer to naive recipients, prime mice for Th2 responsiveness. Heat inactivation of NES abolishes both its ability to drive Th2 responses in vivo and its capacity to stimulate DC for Th2 induction. NES, but not heat-inactivated NES, up-regulates DC maturation markers associated with Th2 promotion (CD86 and OX40L), with little change to CD80 and MHC class II. Moreover, DC exposed to NES readily produce IL-6 and IL-12p40, but not IL-12p70. LPS induced high IL-12p70 levels, except in DC that had been pre-incubated with NES. These data contradict the default hypothesis, demonstrating that a helminth product (NES) actively matures DC, selectively up-regulating CD86 and OX40L together with IL-6 production, while blocking IL-12p70 responsiveness in a manner consistent with Th2 generation in vivo.
Assuntos
Antígenos de Helmintos/imunologia , Células Dendríticas/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Células Th2/imunologia , Animais , Antígenos de Helmintos/metabolismo , Diferenciação Celular/imunologia , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/parasitologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Nippostrongylus/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA/química , RNA/genética , Regulação para Cima/imunologiaRESUMO
BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.