RESUMO
The free-living nematode Caenorhabditis elegans is a tractable experimental model system for the study of both vertebrate and invertebrate biology. Its most significant advantages are its simplicity, both in anatomy and in genomic organization, and the elaborate methods that have been developed to attribute function to previously uncharacterized genes. Importantly, > 40% of parasitic nematode genes exhibit high levels of homology to genes within the C. elegans genome. Studying such genes using the C. elegans model should yield new insights into key molecules and their possible implications in parasite survival, leading to the discovery of new drug targets and vaccine candidates.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Genômica , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Caenorhabditis elegans/efeitos dos fármacos , Transformação Genética/fisiologiaRESUMO
Although the mechanisms underlying the host inflammatory response in ocular onchocerciasis have been examined, the role of particular parasite proteins in this process remains largely unexplored. Recently, it was found that one of the most abundant expressed sequence tags in Onchocerca volvulus infective larvae encoded a protein with similarities to a component of vespid venom. This clone was designated O. volvulus Activation associated Secreted Protein -1 (Ov-asp-1). We report the characterization of three members of a family of proteins, designated the Ov-ASP family, of which Ov-ASP-1 is a member. Sequence based and phylogenetic analyses suggest that these proteins form a filarial specific protein family related to both the vespid venom antigen 5 and the vertebrate CRISP/Tpx family of proteins. The three members of the Ov-ASP family exhibit distinct patterns of expression in the life cycle of O. volvulus. Genomic Southern blot analyses indicate that several genes encoding sequences related to the Ov-asp family are present in the genome of O. volvulus. Recombinant proteins expressed from full length cDNAs encoding two members of the Ov-asp family were found to induce an angiogenic response after injection into corneas of naive mice, and vessel formation was associated with only minor inflammatory cell infiltration. These data suggest that Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratitis.
Assuntos
Antígenos de Helmintos , Proteínas de Helminto/fisiologia , Neovascularização Patológica , Onchocerca volvulus/patogenicidade , Oncocercose Ocular/parasitologia , Venenos de Vespas/genética , Animais , Córnea/irrigação sanguínea , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Onchocerca volvulus/metabolismo , Oncocercose Ocular/patologia , Filogenia , Proteínas Recombinantes de Fusão/farmacologia , Análise de Sequência de DNA , Venenos de Vespas/metabolismoRESUMO
The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Onchocerca volvulus/enzimologia , Estresse Oxidativo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Feminino , Glutationa Transferase/química , Dados de Sequência Molecular , Onchocerca volvulus/fisiologia , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Transcrição GênicaRESUMO
This study describes the histological localization of two CuZn superoxide dismutases (SOD1 and SOD2) in the parasitic nematode Onchocerca volvulus, and a functional characterization of the 'extracellular' form of this enzyme (SOD2) which provides evidence that it is involved in the defense against environmental superoxide anion radicals. These essential enzymes are detected in larval and adult stages of the parasite, determined at the mRNA and protein levels by in situ hybridization and immunolocalization studies. These proteins are distributed throughout the worm, at various concentrations with particularly high levels produced in the hypodermis. In vitro maintenance of parasites indicated that SOD2 was secreted outside the parasite into the medium. Baculovirus constructs designed to test the ability of the SOD2 hydrophobic N-terminal region to function in processing and secretion confirmed the ability of this polypeptide sequence to direct the secretion of a marker protein, as well as of the mature SOD2 enzyme. Analyses of the native, mature SOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the SOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein. The detection of SOD2 outside of the parasite maintained in vitro, and the confirmation that the SOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.
Assuntos
Onchocerca volvulus/enzimologia , Superóxido Dismutase/metabolismo , Animais , Citosol/enzimologia , Escherichia coli/genética , Espaço Extracelular/enzimologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Onchocerca volvulus/genética , Onchocerca volvulus/crescimento & desenvolvimento , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genéticaRESUMO
The genes encoding three forms of superoxide dismutase, the cytosolic and extracellular CuZn superoxide dismutases and the mitochondrial Mn superoxide dismutase, were isolated from an Onchocerca volvulus lambda fix II genomic library. Genomic Southern blot analyses indicate single-copy genes in the O. volvulus genome. The O. volvulus cytosolic and extracellular CuZnSOD genes (Ov-sod-1 and Ov-sod-2) are separated by 0.8 kb of sequence and are convergently transcribed. Since the transcripts from all three sod genes are trans-spliced, the transcription start point of each gene was determined in a heterologous system that lacks trans-splicing machinery by in vitro transcription using Drosophila embryo nuclear extracts, followed by primer extension experiments. The ability of the 5' flanking region of the genes encoding the three Ov-SODs to promote transcription was further examined in transient transfections of Chinese hamster ovary cells. In firefly luciferase reporter assays, the Ov-sod-1 and -2 and the MnSOD (Ov-sod-3) gene promoters showed minimal, strong, and moderate levels of activity in these cells, respectively. Both Ov-sod-2 and -3 gene promoter regions showed an initial increase in activity in response to 5' deletions. The results from the in vitro transcription experiments and the luciferase reporter assays were consistent and suggest the presence of Inr-like elements in the promoter regions of the Ov-sod genes.
Assuntos
Onchocerca volvulus/genética , Regiões Promotoras Genéticas , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Animais , Sequência de Bases , Southern Blotting , Células CHO , Cricetinae , DNA de Helmintos/química , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Genoma , Luciferases/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Onchocerca volvulus/enzimologia , RNA de Helmintos/metabolismo , Mapeamento por Restrição , Transcrição Gênica , TransfecçãoRESUMO
In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans . A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls. Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stress-responsive protein. Since paraquat is known to generate superoxide radicals in vivo , the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations did not show any apparent increase in the levels of these SOD mRNAs.
Assuntos
Caenorhabditis elegans/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Helmintos , RNA Mensageiro/biossíntese , Fatores de Transcrição/biossíntese , Transcrição Gênica , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , DNA Complementar , Glutationa Transferase/biossíntese , Larva , Zíper de Leucina , Dados de Sequência Molecular , Paraquat/farmacologia , Reação em Cadeia da Polimerase/métodos , RNA de Helmintos/biossíntese , Estresse Fisiológico , Superóxido Dismutase/biossíntese , Fatores de Transcrição/química , Dedos de ZincoRESUMO
The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5'-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5'-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5' genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.
Assuntos
Genes de Helmintos , Onchocerca volvulus/genética , Superóxido Dismutase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/química , DNA Complementar/genética , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Vaccination of mice with a recombinant protein, Ov-ASP-1, the Onchocerca volvulus homologue of the activation associated secreted gene family stimulated very high titres of both IgG1 and IgG2a without adjuvant. rOv-ASP-1 was also immuno-reactive with IgG isotypes from both O. volvulus-infected (INF) and putatively immune (PI) humans, with higher IgG4 in the former group. The protein also stimulated IFN-gamma secretion by PBMC from INF and PI and IL-5 only in INF. Using a mouse diffusion chamber model, vaccination with rOv-ASP-1 resulted in partial but significant protection against challenge with infective third-stage larvae (L3) but only when formulated with Freund's complete adjuvant (FCA) or alum. Protection was Th1-dependent (highly elevated IgG2a) with FCA and contingent on a strongly Th2-skewed (IgG1) response with alum. IgE responses to rOv-ASP-1 with or without adjuvant were weak or absent. When immunization using rOv-ASP-1 in adjuvant failed to induce adequate Th1 (FCA) or Th2 (alum) responses, protection efficacy was compromised. The recombinant protein appears to stimulate a mixed Th1/Th2 response but the outcome in terms of protective immunity is the result of a subtle interplay of its intrinsic and adjuvant-augmented properties. Ov-ASP-1 is potentially secreted based on its localization in the secretory granules of L3.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Onchocerca volvulus/imunologia , Oncocercose/imunologia , Oncocercose/prevenção & controle , Adjuvantes Imunológicos , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Antígenos de Helmintos/metabolismo , Técnicas de Cultura de Células , Esôfago/metabolismo , Adjuvante de Freund , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interferon gama/análise , Interferon gama/biossíntese , Interleucina-5/análise , Interleucina-5/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oncocercose/parasitologia , Proteínas Recombinantes/imunologia , Vesículas Secretórias/metabolismo , VacinaçãoRESUMO
The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment of Onchocerca volvulus infection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. Other O. volvulus genes showed homology only to predicted genes from the free-living nematode Caenorhabditis elegans or were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology of O. volvulus as well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research.