RESUMEN
RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.
RESUMEN
A closed-tube real-time PCR (RT PCR) method was developed to identify individual strongylid nematode larvae recovered from ovine faecal cultures. The method builds on an earlier conventional PCR assay established by our group and similarly targets species-specific sequence motifs in the ITS-2 region of ribosomal DNA. The new procedure combines RT PCR with DNA melting curve analyses to identify species-specific amplicons, thus avoiding the need to undertake gel electrophoresis. As with the earlier method, it involves two sets of species-specific reactions. The first targets Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum venulosum while the second targets Trichostrongylus axei, Trichostrongylus vitrinis, Cooperia curticei and Chabertia ovina. With two exceptions, all the DNA primers employed in the new assay were among those described and tested in developing the earlier assay. The exceptions are the forward "generic" primer, which was re-designed to generate smaller amplicon sizes more suitable for melting curve analyses, and the T. axei-specific primer, which was modified to achieve a higher amplicon melt temperature to enable larvae of this species to be more readily differentiated from those of C. curticei. The melt temperature range for amplicons representing each of the species targeted was determined using lysates derived from both microscopically identified adult male worms (2-12/species), as well as 30 larvae of each of the species which were derived from at least 6 different geographical locations throughout New Zealand. The new assay potentially provides a simpler, faster method to identify individual ovine strongylid larvae for downstream applications than was provided by the earlier conventional PCR assay.
Asunto(s)
Parasitología/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Strongylida/parasitología , Estrongílidos/genética , Animales , ADN Espaciador Ribosómico/genética , Heces/parasitología , Larva , Masculino , Nueva Zelanda , Desnaturalización de Ácido Nucleico , Ovinos , Especificidad de la Especie , Estrongílidos/clasificaciónRESUMEN
Full length cDNA encoding ornithine decarboxylases (ODC; EC 4.1.1.17) were cloned from the sheep abomasal nematode parasites Teladorsagia circumcincta (TcODC) and Haemonchus contortus (HcODC). The TcODC (1272 bp) and HcODC cDNA (1266 bp) encoded 424 and 422 amino acid proteins respectively. The predicted TcODC amino acid sequence showed 87% identity with HcODC and 65% and 64% with Caenorhabditis elegans and Caenorhabditis briggsae ODC respectively. All binding sites and active regions were completely conserved in both proteins. Soluble N-terminal His-tagged ODC proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcODC and HcODC had very similar kinetic properties: K(m) ornithine was 0.2-0.25 mM, optimum [PLP] was 0.3 mM and the pH optima were pH 8. No enzyme activity was detected when arginine was used as substrate. One millimolar difluoromethylornithine (DFMO) completely inhibited TcODC and HcODC activity, whereas 2 mM agmatine did not inhibit activity. The present study showed that ODC is a separate enzyme from arginine decarboxylase and strictly uses ornithine as substrate.
Asunto(s)
Haemonchus/enzimología , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Ovinos/parasitología , Trichostrongyloidea/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Haemonchus/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Trichostrongyloidea/genéticaRESUMEN
The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD(+)/H or NADP(+)/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD(+)/H than with NADP(+)/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.
Asunto(s)
Glutamato Deshidrogenasa/metabolismo , Haemonchus/enzimología , Nucleótidos/farmacología , Trichostrongyloidea/enzimología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutamato Deshidrogenasa/genética , Guanosina Trifosfato/farmacología , Haemonchus/efectos de los fármacos , Haemonchus/genética , Concentración de Iones de Hidrógeno/efectos de los fármacos , Cinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Trichostrongyloidea/efectos de los fármacosRESUMEN
BACKGROUND: Gastrointestinal nematode (GIN) infections are the predominant cause of economic losses in sheep. Infections are controlled almost exclusively by the use of anthelmintics which has lead to the selection of drug resistant nematode strains. An alternative control approach would be the induction of protective immunity to these parasites. This study exploits an ovine microarray biased towards immune genes, an artificially induced immunity model and the use of pseudo-afferent lymphatic cannulation to sample immune cells draining from the intestine, to investigate possible mechanisms involved in the development of immunity. RESULTS: During the development of immunity to, and a subsequent challenge infection with Trichostrongylus colubriformis, the transcript levels of 2603 genes of cells trafficking in afferent intestinal lymph were significantly modulated (P < 0.05). Of these, 188 genes were modulated more than 1.3-fold and involved in immune function. Overall, there was a clear trend for down-regulation of many genes involved in immune functions including antigen presentation, caveolar-mediated endocytosis and protein ubiquitination. The transcript levels of TNF receptor associated factor 5 (TRAF5), hemopexin (HPX), cysteine dioxygenase (CDO1), the major histocompatability complex Class II protein (HLA-DMA), interleukin-18 binding protein (IL-18BP), ephrin A1 (EFNA1) and selenoprotein S (SELS) were modulated to the greatest degree. CONCLUSIONS: This report describes gene expression profiles of afferent lymph cells in sheep developing immunity to nematode infection. Results presented show a global down-regulation of the expression of immune genes which may be reflective of the natural temporal response to nematode infections in livestock.
Asunto(s)
Regulación hacia Abajo/inmunología , Enfermedades Gastrointestinales/inmunología , Linfa/metabolismo , Tricostrongiliasis/inmunología , Trichostrongylus/inmunología , Animales , Cateterismo , Cisteína-Dioxigenasa/genética , Cisteína-Dioxigenasa/inmunología , Cisteína-Dioxigenasa/metabolismo , Enfermedades Gastrointestinales/genética , Enfermedades Gastrointestinales/parasitología , Perfilación de la Expresión Génica , Hemopexina/genética , Hemopexina/inmunología , Hemopexina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/parasitología , Intestinos/patología , Linfa/citología , Linfa/inmunología , Linfa/parasitología , Análisis por Matrices de Proteínas , Ovinos , Factor 5 Asociado a Receptor de TNF/genética , Factor 5 Asociado a Receptor de TNF/inmunología , Factor 5 Asociado a Receptor de TNF/metabolismo , Tricostrongiliasis/genética , Tricostrongiliasis/parasitologíaRESUMEN
Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Glucosafosfato Deshidrogenasa/metabolismo , Nicotiana/metabolismo , Secuencia de Bases , Cloroplastos/enzimología , ADN sin Sentido/genética , ADN de Plantas/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucosafosfato Deshidrogenasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Estrés Oxidativo , Fenotipo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
Bacillus thuringiensis that produce Cry1Ba are toxic to Lucilia cuprina Wiedemann blow fly maggots in vivo, and when applied in quantity to sheep fleece, provide up to 6 wk protection against flystrike in the field. These strains also are toxic to Epiphyas postvittana (Walker) light brown apple moth caterpillars. B. thuringiensis expressing Cry1Db are toxic only to E. postvittana. When Cry1Ba and Cry1Db proteins are expressed within Escherichia coli, the recombinant bacteria have the same toxicity profile as the wild-type B. thuringiensis strain. In an effort to develop a Cry protein with improved blow fly toxicity, three different internal regions of Cry1Ba coding DNA, encoding all or part of domains I, II and III respectively were systematically exchanged with the corresponding region from a pool of other Cry protein coding DNAs. The chimeric products were then expressed in recombinant E. coli, and the resulting bacteria assayed for toxicity on L. cuprina and E. postvittana. Clones having insecticide bioactivity were characterized to identify the source of the replacement Cry domain. Despite successfully expressing a large number and variety of chimeric proteins within E. coli, many with measurable insecticidal activity, none of the chimeras had greater potency against L. cuprina than the wild-type Cry1Ba. Chimeric replacements involving domains I and II were rarely active, whereas a much higher proportion of domain III chimeras had some bioactivity. We conclude that shuffling of Cry coding regions through joining at the major conserved sequence motifs is an effective means for the production of a diverse number of chimeric Cry proteins but that such toxins with enhanced bioactive properties will be rare or nonexistent.