Hc-daf-2 encodes an insulin-like receptor kinase in the barber's pole worm, Haemonchus contortus, and restores partial dauer regulation
- Li, Facai, Lok, James, Gasser, Robin, Korhonen, Pasi, Sandeman, Mark, Shi, Deshi, Zhou, Rui, Li, Xiangrui, Zhou, Yanqin, Zhao, Junlong, Hu, Min
- Authors: Li, Facai , Lok, James , Gasser, Robin , Korhonen, Pasi , Sandeman, Mark , Shi, Deshi , Zhou, Rui , Li, Xiangrui , Zhou, Yanqin , Zhao, Junlong , Hu, Min
- Date: 2014
- Type: Text , Journal article
- Relation: International Journal for Parasitology Vol. 44, no. 7 (2014), p. 485-496
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- Description: Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/. Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism. © 2014 Australian Society for Parasitology Inc.
- Authors: Li, Facai , Lok, James , Gasser, Robin , Korhonen, Pasi , Sandeman, Mark , Shi, Deshi , Zhou, Rui , Li, Xiangrui , Zhou, Yanqin , Zhao, Junlong , Hu, Min
- Date: 2014
- Type: Text , Journal article
- Relation: International Journal for Parasitology Vol. 44, no. 7 (2014), p. 485-496
- Full Text:
- Reviewed:
- Description: Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/. Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism. © 2014 Australian Society for Parasitology Inc.
Low cost whole-organism screening of compounds for anthelmintic activity
- Preston, Sarah, Jabbar, Abdul, Nowell, Cameron, Joachim, Anja, Ruttkowski, Barbel, Baell, Jonathan, Cardno, Tony, Korhonen, Pasi, Piedrafita, David, Ansell, Brendan, Jex, Aaron, Hofmann, Andreas, Gasser, Robin
- Authors: Preston, Sarah , Jabbar, Abdul , Nowell, Cameron , Joachim, Anja , Ruttkowski, Barbel , Baell, Jonathan , Cardno, Tony , Korhonen, Pasi , Piedrafita, David , Ansell, Brendan , Jex, Aaron , Hofmann, Andreas , Gasser, Robin
- Date: 2015
- Type: Text , Journal article
- Relation: International Journal for Parasitology Vol. 45, no. 5 (2015), p. 333-343
- Full Text: false
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- Description: Due to major problems with drug resistance in parasitic nematodes of animals, there is a substantial need and excellent opportunities to develop new anthelmintics via genomic-guided and/or repurposing approaches. In the present study, we established a practical and cost-effective whole-organism assay for the in vitro-screening of compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). The assay is based on the use of exsheathed L3 (xL3) and L4 stages of H. contortus of small ruminants (sheep and goats). Using this assay, we screened a panel of 522 well-curated kinase inhibitors (GlaxoSmithKline, USA; code: PKIS2) for activity against H. contortus by measuring the inhibition of larval motility using an automated image analysis system. We identified two chemicals within the compound classes biphenyl amides and pyrazolo[1,5-α]pyridines, which reproducibly inhibit both xL3 and L4 motility and development, with IC50s of 14-47μM. Given that these inhibitors were designed as anti-inflammatory drugs for use in humans and fit the Lipinski rule-of-five (including bioavailability), they show promise for hit-to-lead optimisation and repurposing for use against parasitic nematodes. The screening assay established here has significant advantages over conventional methods, particularly in terms of ease of use, throughput, time and cost. Although not yet fully automated, the current assay is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation. The current assay is highly adaptable to many parasites of socioeconomic importance, including those causing neglected tropical diseases. This aspect is of major relevance, given the urgent need to deliver the goals of the London Declaration (
Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour
- Kumarasingha, Rasika, Karpe, Avinash, Preston, Sarah, Yeo, Tiong-Chia, Lim, Diana, Tu, Chu-Lee, Luu, Jennii, Simpson, Kaylene, Shaw, Jillian, Gasser, Robin, Beale, David, Morrison, Paul, Palombo, Enzo, Boag, Peter
- Authors: Kumarasingha, Rasika , Karpe, Avinash , Preston, Sarah , Yeo, Tiong-Chia , Lim, Diana , Tu, Chu-Lee , Luu, Jennii , Simpson, Kaylene , Shaw, Jillian , Gasser, Robin , Beale, David , Morrison, Paul , Palombo, Enzo , Boag, Peter
- Date: 2016
- Type: Text , Journal article
- Relation: International Journal for Parasitology: Drugs and Drug Resistance Vol. 6, no. 3 (2016), p. 171-178
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- Description: Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented. Image 1 •Chemical fractionation of Picria fel-terrae Lour. plant extract.•Anthelmintic activity against Caenorhabditis elegans and Haemonchus contortus.•Metabolic profiling and chemometric analysis of active fraction.•Active fraction has minimal mammalian cytotoxicity.
- Authors: Kumarasingha, Rasika , Karpe, Avinash , Preston, Sarah , Yeo, Tiong-Chia , Lim, Diana , Tu, Chu-Lee , Luu, Jennii , Simpson, Kaylene , Shaw, Jillian , Gasser, Robin , Beale, David , Morrison, Paul , Palombo, Enzo , Boag, Peter
- Date: 2016
- Type: Text , Journal article
- Relation: International Journal for Parasitology: Drugs and Drug Resistance Vol. 6, no. 3 (2016), p. 171-178
- Full Text:
- Reviewed:
- Description: Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented. Image 1 •Chemical fractionation of Picria fel-terrae Lour. plant extract.•Anthelmintic activity against Caenorhabditis elegans and Haemonchus contortus.•Metabolic profiling and chemometric analysis of active fraction.•Active fraction has minimal mammalian cytotoxicity.
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