Nematode Haemonchus Contortus Research Articles

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

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.

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15mg/kg), or nine times the recommended dose rate (45mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)200 SNP and a decrease in the (TAC)167 SNP. All worms genotyped were GAA at codon 198.

In vitro screening of plant lectins and tropical plant extracts for anthelmintic properties

Lectins are plant secondary metabolites (PSM) found in many forages and which may confer anthelmintic properties to gastrointestinal parasites through disrupting the development of parasitic larvae throughout its life cycle. In experiment 1, the ability of the plant lectins jacalin (JAC), concanavalin A (Con A), phytohemagglutinin E2L2 (PHA-E2L2), phytohemagglutinin L4 (PHA-L4), phytohemagglutinin E3L (PHA-E3L), kidney bean albumin (KBA), Robinia pseudoacacia agglutinin (RPA), Maackia amurensis lectin (MAA), Maclura pomifera agglutinin (MAA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) to disrupt the feeding of the first stage larvae (L1) of the sheep gastro-intestinal nematodes (GIN) Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis was investigated using a larval feeding inhibition test (LFIT). Only PHA-E3L, WGA and Con A had a potent effect on disrupting larval feeding of all of the three species of GIN investigated. The lectin concentration required to inhibit feeding in 50% of L1 (IC50) was 7.3±1.2, 8.3±1.4 and 4.3±1.7μg/ml for PHA-E3L; 59.1±32.4, 58.7±11.9 and 8.1±7.0μg/ml for Con A and 78.9±11.2, 69.4±8.1 and 28.0±14.1μg/ml for WGA for T. circumcincta, H. contortus and T. colubriformis larvae, respectively (P=0.006). The addition of the lectin inhibitors fetuin, glucose/mannose or N-acetylglucosamine for PHA-E3L, Con A and WGA, respectively, caused an increase in the proportion of larvae that had fed at all concentrations for PHA-E3L only. In experiment 2, the effect of extracts from the tropical plants Azadiractha indica, Trichanthera gigantea, Morus alba, Gliricidia sepium and Leucaena leucocephala on the feeding behaviour of H. contortus L1, was examined. A. indica, T. gigantea and M. alba failed to inhibit 50% of larvae from feeding at concentrations up to 10mg plant extract per ml. In contrast, both G. sepium and L. leucocephala demonstrated a dose-dependent effect on larval feeding with respective IC50 estimates (mean±s.e.) of 0.015mg/ml±0.001 and 3.465mg/ml±0.144, effects which were partly reversed by the inclusion of either the tannin inhibitor polyethylene glycol or the lectin inhibitor Fetuin. These studies demonstrate that plant lectins can have an inhibitory effect on the feeding behaviour of first stage larvae of ovine GIN in vitro. Moreover they also provide novel evidence that lectins may contribute to the anthelmintic properties of some tropical forage plant extracts, such as G. sepium and L. leucocephala.

Thermodynamic Evaluation of Ligand Binding in the Plant-like Phosphoethanolamine Methyltransferases of the Parasitic Nematode Haemonchus contortus

Nematodes are a major cause of disease and the discovery of new pathways not found in hosts is critical for development of therapeutic targets. Previous studies suggest that Caenorhabditis elegans synthesizes phosphocholine via two S-adenosylmethionine (AdoMet)-dependent phosphoethanolamine methyltransferases (PMT). Here we examine two PMT from the parasitic nematode Haemonchus contortus. Sequence analysis suggests that HcPMT1 contains a methyltransferase domain in the N-terminal half of the protein and that HcPMT2 encodes a C-terminal methyltransferase domain, as in the C. elegans proteins. Kinetic analysis demonstrates that HcPMT1 catalyzes the conversion of phosphoethanolamine to phosphomonomethylethanolamine (pMME) and that HcPMT2 methylates pMME to phosphodimethylethanolamine (pDME) and pDME to phosphocholine. The IC(50) values for miltefosine, sinefungin, amodiaquine, diphenhydramine, and tacrine suggest differences in the active sites of these two enzymes. To examine the interaction of AdoMet and S-adenosylhomocysteine (AdoCys), isothermal titration calorimetry confirmed the presence of a single binding site in each enzyme. Binding of AdoMet and AdoCys is tight (K(d) ∼2-25 μm) over a range of temperatures (5-25 °C) and NaCl concentrations (0.05-0.5 m). Heat capacity changes for AdoMet and AdoCys binding suggests that each HcPMT differs in interaction surface area. Nonlinear van't Hoff plots also indicate a possible conformational change upon AdoMet/AdoCys binding. Functional analysis of the PMT from a parasitic nematode provides new insights on inhibitor and AdoMet/AdoCys binding to these enzymes.

Pharmacological characterization of the Haemonchus contortus GABA-gated chloride channel, Hco-UNC-49: Modulation by macrocyclic lactone anthelmintics and a receptor for piperazine

Invertebrate ligand-gated chloride channels are well recognized as important targets for several insecticides and anthlemintics. Hco-UNC-49 is a GABA-gated chloride channel from the parasitic nematode Haemonchus contortus and is an orthologue to the neuromuscular receptor (Cel-UNC-49) from the free-living nematode Caenorhabditis elegans. While the receptors from the two nematodes are similar in sequence, they exhibit different sensitivities to GABA which may reflect differences in in vivo function. The aim of the current study was to further characterize the pharmacology of the Hco-UNC-49 receptor by examining its sensitivity to various insecticides and anthelmintics using two-electrode voltage clamp. Specifically, the insecticides fipronil and picrotoxin appear to inhibit the channel in a similar manner. The IC50 of picrotoxin on the homomeric channel was 3.65±0.64μM and for the heteromeric channel was 134.56±44.12μM. On the other hand, dieldrin, a well-known insect GABA receptor blocker, had little effect on the UNC-49 channel. The anthelmintics ivermectin and moxidectin both moderately potentiated the activation of Hco-UNC-49 by GABA, while piperazine was able to directly activate both the Hco-UNC-49 homomeric and heteromeric channels with EC50 values of 6.23±0.45mM and 5.09±0.32mM, respectively. This piperazine current was reversibly blocked by picrotoxin which demonstrates that the anthelmintic specifically targets Hco-UNC-49. These results demonstrate that Hco-UNC-49 exhibits binding sites for several molecules including piperazine and macrocyclic lactone anthelmintics. In addition, this is the first report of the heterologous expression and subsequent characterization of a receptor for piperazine.

The influence of water on the migration of infective trichostrongyloid larvae onto grass

Detailed knowledge of the effects of water on the migration of infective larvae of economically important trichostrongyloid species is urgently needed to feed into prediction models of future epidemiology. The influence of water on the migration of the parasitic nematodes Nematodirus battus, Haemonchus contortus and Teladorsagia circumcincta from sheep dung onto grass was examined in a series of laboratory experiments. Turf plots were seeded with larvae, which were recovered from grass clippings by serial sampling. Free water was necessary for larvae to escape from dung, but not for vertical migration onto grass. When temperature and relative humidity were held constant, the proportion of a population of live larvae present on herbage reached a plateau of around 2 (1-10)% after 24 h, and then changed little over time. Larvae in soil and dung formed a reservoir, such that a similar proportion of the larval population was maintained on grass after clipping. These findings suggest continuing random movement of free larvae. Implications for the epidemiology of trichostrongyloid species are discussed in the context of trade-offs faced by the parasites.

The Haemonchus contortus UNC-49B subunit possesses the residues required for GABA sensitivity in homomeric and heteromeric channels

Hco-UNC-49 is a GABA receptor from the parasitic nematode Haemonchus contortus that has a relatively low overall sequence similarity to vertebrate GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. While the nematode receptors do share >80% sequence similarity they exhibit different sensitivities to GABA. In addition, the UNC-49C subunit appears to be a positive modulator of GABA sensitivity in the H. contortus heteromeric channel, but is a negative modulator in the C. elegans heteromeric channel. The cause(s) of these differences is currently unknown since the structural elements essential for GABA sensitivity in nematode receptors have been largely unexplored. Thus, the overall aim of this study was to investigate the residues that are important for UNC-49 receptor sensitivity through the use of homology modeling, site-directed mutagenesis, and two-electrode voltage clamp. This study revealed that Met 170 in Loop B of the GABA binding-site may partially account for the observed differences in GABA receptor sensitivity between the nematode species. Residues in Loops A–D that have been reported to form the GABA binding pocket in mammalian receptors, including those forming the conserved ‘aromatic box’, also appear to play analogous roles in Hco-UNC-49. In addition, the two mutations that produced the most significant reduction in GABA sensitivity were R66S and Y166S. Homology modeling indicates that these two residues share a hydrogen bond and are positioned close to the carboxyl end of the GABA molecule. However, of residues examined in this study, only those on the Hco-UNC-49B subunit and not its subunit partner, Hco-UNC-49C, appear important for GABA sensitivity. Overall, results from this study suggest that the binding site of the UNC-49 heteromeric GABA receptor exhibits some differences compared to classical vertebrate GABA A receptors.

The activity and hydrogen peroxide sensitivity of the peroxiredoxins from the parasitic nematode Haemonchus contortus

The requirement of aerobic organisms to control damage caused by reactive oxygen species has led to the evolution of the antioxidant systems. Peroxiredoxins are a large family of peroxidases which detoxify hydrogen peroxide at the expense of thiols. The parasitic nematode Haemonchus contortus contains two peroxiredoxins, HcPrx1 a mitochondrial protein and HcPrx2 a cytoplasmic protein. Although both peroxiredoxins contain the conserved eukaryotic motifs ‘GGLG’ and ‘YF’, identified as critical for hydrogen peroxide instability, both were stable to high concentrations of hydrogen peroxide, demonstrating different functions to their mammalian counterparts. H. contortus also contains two thioredoxin reductases and five different thioredoxin-like proteins. The activity of both peroxiredoxins was specific for the thioredoxin system; however, both could also be regenerated by the glutathione system when coupled to the nematode specific thioredoxin HcTrx5. Analysis of homologous genes in Caenorhabditis elegans showed that only CePrx2, which is secreted, was sensitive to the external oxidant hydrogen peroxide. However, both peroxiredoxins KO C. elegans were sensitive to intracellular free radicals and both peroxiredoxins protected DNA from free radical attack. The results demonstrate that the hydrogen peroxide detoxification and the antioxidant activity of the peroxiredoxins are separate activities that are independent of the ‘GGLG’ and ‘YF’ motifs.

Factors affecting susceptibility to RNA interference in Haemonchuscontortus and in vivo silencing of an H11 aminopeptidase gene

Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of β-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, β-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites.

A suite of genes expressed during transition to parasitic lifestyle in the trichostrongylid nematode Haemonchus contortus encode potentially secreted proteins conserved in Teladorsagia circumcincta

The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, however spreading of anthelmintic resistance has reduced their efficacy. The genes involved in the transition to parasitic lifestyle could constitute targets of interest to develop alternative control strategies. In the trichostrongylid nematode Haemonchus contortus, we have used a SSH (Suppressive Subtractive Hybridization) based approach to generate two distinct subtracted cDNA libraries specifically enriched in cDNA expressed during the early parasitic fourth stage larvae L4 (5 days post-infection). A total of 200 clones were subjected to dot-blot experiments and 46 clones were selected for further characterization. The 46 corresponding expressed sequence tags (EST) were found to cluster into nine contigs. The corresponding full-length cDNA was obtained for all candidates. The genes encoding potentially secreted proteins were investigated in more detail. RT-PCR experiments confirmed their specific expression or over expression from the early L4 larvae to the adult stages and search for homologs in the trichostrongylid species T. circumcincta was performed in order to investigate whether they may be novel cross-specific targets.

Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematodeHaemonchus contortus

In nematodes as in other eukaryotes, there is increasing evidence that drug resistance depends on both changes in the drug cellular targets and in nonspecific mechanisms, involving cellular detoxification by efflux pumps. In vertebrates, P-glycoproteins (Pgp) are membrane efflux pumps responsible for the elimination of xenobiotic agents, especially drugs. We previously reported the presence of Pgp pumps in eggshells and cuticles of the nematode Haemonchus contortus. Eggshells and cuticles are different from cell membranes, in particular they include a chitin layer. Nevertheless these structures present some common biological features with cell membranes and play a role in xenobiotic transport. Pgp activity has been shown to depend on the lipid environment and, in particular, on the cholesterol content in both vertebrate and nematode models. In vertebrates, Pgp is in part located in membrane cholesterol-enriched microdomains, the rafts. We describe here, for the first time, lipid microdomains in eggshells that could correspond with raft-like structures (RLSs). Moreover, a large proportion of the Pgp was shown to colocalize with these RLSs. The functional consequences of the colocalization for xenobiotic transport and thus drug resistance in nematodes were analyzed and compared with results obtained in vertebrates. An understanding of such mechanisms is crucial in overcoming the failure of drug treatments due to the development of resistance.

Substrate specificity of the mitochondrial thioredoxin reductase of the parasitic nematode Haemonchus contortus

Thioredoxin reductase (TrxR) is an evolutionary diverse enzyme. In higher eukaryotes, TrxR contains a selenocysteine in the active site which confers high activity and a broad substrate range. The parasitic nematode Haemonchus contortus contains two TrxRs, a cytoplasmic enzyme HcTrxR1 with a selenocysteine in the active site (Gly-Cys-SeCys-Gly), similar to the mammalian TrxR, and a mitochondrial enzyme HcTrxR2 with a Gly-Cyc-Cys-Gly active site which is unique to nematodes. Both enzymes were cloned and expressed and their activity compared to rat TrxR. Although the expressed HcTrxR1 contained a cysteine to replace the selenocycteine, it showed broad activity with thioredoxins from Escherichia coli, sheep, and H. contortus similar to the rat selenoenzyme. However, HcTrxR2 had high activity only with the mitochondrial H. contortus thioredoxin. HcTrxR2 was also active with the commonly used substrate 5,5'-dithiobis(2-nitrobenzoate) and sodium selenite and was inhibited by auranofin and the natural product compounds curcumin and theaflavin. This demonstrates that the non-seleno-enzyme HcTrxR2 is as active as the rat seleno-containing enzyme when reacting with the H. contortus thioredoxin.

An UNC‐49 GABA receptor subunit from the parasitic nematode Haemonchus contortus is associated with enhanced GABA sensitivity in nematode heteromeric channels

We have identified two genes from the parasitic nematode Haemonchus contortus, Hco-unc-49B and Hco-unc-49C that encode two GABA-gated chloride channel subunits. Electrophysiological analysis revealed that this channel has properties similar to those of the UNC-49 channel from the free-living nematode Caenorhabditis elegans. For example, the Hco-UNC-49B subunit forms a functional homomeric channel that responds to GABA and is highly sensitive to picrotoxin. Hco-UNC-49C alone does not respond to GABA but can assemble with Hco-UNC-49B to form a heteromeric channel with a lower sensitivity to picrotoxin. However, we did find that the Hco-UNC-49B/C heteromeric channel is significantly more responsive to agonists compared to the Hco-UNC-49B homomeric channel, which is the opposite trend to what has been found previously for the C. elegans channel. To investigate the subunit requirements for high agonist sensitivity, we generated cross-assembled channels by co-expressing the H. contortus subunits with UNC-49 subunits from C. elegans (Cel-UNC-49). Co-expressing Cel-UNC-49B with Hco-UNC-49C produced a heteromeric channel with a reduced sensitivity to GABA compared to that of the Cel-UNC-49B homomeric channel. In contrast, co-expressing Hco-UNC-49B with Cel-UNC-49C produced a heteromeric channel that, like the Hco-UNC-49B/C heteromeric channel, exhibits an increased sensitivity to GABA. These results suggest that the Hco-UNC-49B subunit is the key determinant for the high agonist sensitivity of heteromeric channels.

A vacuolar-type proton (H +) translocating ATPase α subunit encoded by the Hc- vha- 6 gene of Haemonchus contortus

In the present study, a full-length cDNA (designated Hc- vha- 6) inferred to encode an α subunit of a vacuolar-type proton translocating adenosine triphosphatase (V-ATPase) was isolated from the parasitic nematode Haemonchus contortus, and characterized. The transcript for Hc- vha- 6 was detected in all developmental stages and both sexes of H. contortus. Elements, including two TATA box (TATAA), two inverted CAAT box (ATTGG), five E box (CANNTG) and six GATA as well as five inverse GATA (TTATC) transcription factor motifs, were identified in the non-coding region upstream of Hc- vha- 6. The open reading frame (ORF) of 2601 nucleotides encoded a protein ( Hc-VHA-6) of 866 amino acids and a molecular weight of ∼98.7 kDa. Comparison with a published protein sequence for a homologue (VPH1P) from yeast showed that Hc-VHA-6 had nine transmembrane domains and the 14 essential amino acid residues associated with enzyme activity, assembly, intracellular and/or membrane targeting. Phylogenetic analyses of selected amino acid sequence data revealed Hc-VHA-6 to be most closely related to VHA-6 of Caenorhabditis elegans. A predictive network analysis inferred that vha- 6 interacts with at least seven other genes encoding V-ATPase subunits and a small Rab GTPase. This study provides the first insight into a V-ATPase of parasitic nematodes and a sound basis for future functional genomic work.

Protein acyltransferase function of purified calreticulin. Part 1: characterization of propionylation of protein utilizing propoxycoumarin as the propionyl group donor

We have earlier reported that an endoplasmic reticulum luminal protein calreticulin (CR) mediated the acetylation of certain receptor proteins such as glutathione S-transferase (GST) by polyphenolic acetates, leading to irreversible inhibition. This function of calreticulin was termed calreticulin transacetylase. In this communication, we have demonstrated for the first time the ability of the purified recombinant calreticulin of a parasitic nematode Haemonchus contortus to transfer propionyl group from 7,8-Dipropoxy-4-methylcoumarin (DPMC) to recombinant Schistosoma japonicum glutathione S-transferase (rGST). Calreticulin transacetylase exhibited hyperbolic kinetics and yielded K(m) (140 microM) and V(max) (105 units) when the concentration of DPMC was varied keeping the concentration of rGST constant. rGST thus propionylated was found to positively interact with anti-acetyl lysine antibody. Also, the nanoscale LC-MS/MS analysis identified the propionylation sites on three lysine residues: Lys-11, -180 and -181 of rGST. These results highlight the transacylase function of calreticulin (CRTAase).

Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23).

Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 μg of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

Design and Synthesis of Novel Insecticides Based on the Serotonergic Ligand 1-[(4-Aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine (PAPP)

1-[(4-Aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine (PAPP) is a 5-HT(1A) agonist and was reported to display high affinity for serotonin (5-HT) receptor from the parasitic nematode Haemonchus contortus . The present investigation explored the possibility of using PAPP as a lead compound of new insecticides with novel mode of action. On the basis of the PAPP scaffold, a series of 1-arylmethyl-4-[(trifluoromethyl)pyridin-2-yl]piperazine derivatives were designed, synthesized, and evaluated for biological activities against the armyworm Pseudaletia separata (Walker). Bioassays showed that most of the target compounds displayed certain growth-inhibiting activities or larvicidal activities against armyworm. The quantitative structure-activity relationship (QSAR) for growth-inhibiting activities was also analyzed and established.

Structural and functional characterisation of the fork head transcription factor-encoding gene, Hc-daf-16, from the parasitic nematode Haemonchus contortus (Strongylida)

Despite their phylogenetic diversity, parasitic nematodes share attributes of longevity and developmental arrest (=hypobiosis) with free-living nematodes at key points in their life cycles, particularly in larval stages responsible for establishing infection in the host. Insulin-like signalling plays crucial roles in the regulation of life span and arrest (=dauer formation) in the free-living nematode, Caenorhabditis elegans. Insulin-like signalling in C. elegans negatively regulates the fork head boxO (FoxO) transcription factor encoded by daf-16, which is linked to initiating a dauer-specific pattern of gene expression. Orthologues of daf-16 have been identified in several species of parasitic nematode. Although function has been demonstrated for an orthologue from the parasitic nematode Strongyloides stercoralis (Rhabditida), the functional capabilities of homologues/orthologues in bursate nematodes (Strongylida) are unknown. In the present study, we used a genomic approach to determine the structures of two complete daf-16 orthologues (designated Hc-daf-16.1 and Hc-daf-16.2) and their transcripts in the parasitic nematode Haemonchus contortus, and assessed their function(s) using C. elegans as a genetic surrogate. Unlike the multiple isoforms of Ce-DAF-16 and Ss-DAF-16, which are encoded by a single gene and produced by alternative splicing, mRNAs encoding the proteins Hc-DAF-16.1 and Hc-DAF-16.2 are transcribed from separate and distinct loci. Both orthologues are transcribed in all developmental stages and both sexes of H. contortus, and the inferred proteins (603 and 556 amino acids) each contain a characteristic, highly conserved fork head domain. In spite of distinct differences in genomic organisation compared with orthologues in C. elegans and S. stercoralis, genetic complementation studies demonstrated here that Hc-daf-16.2, but not Hc-daf-16.1, could restore daf-16 function to a C. elegans strain carrying a null mutation at this locus. These findings are consistent with previous results for S. stercoralis and demonstrate functional conservation of the daf-16b orthologue between key parasitic nematodes from two different taxonomic orders and C. elegans. We conclude from these experiments that the fork head transcription factor DAF-16 and, by inference, other insulin-like signalling elements, are conserved in H. contortus, a parasitic nematode of paramount economic importance. We demonstrate that functionality is sufficiently conserved in Hc-DAF-16.2 that it can replace Ce-DAF-16 in promoting dauer arrest in C. elegans.

FMRFamide-like neuropeptides as putative ligands of the latrophilin-like HC110-R from Haemonchus contortus

The latrophilin-like receptor HC110-R of the parasitic nematode Haemonchus contortus has been previously identified as a target for the novel anthelmintic drug emodepside, but the natural ligand(s) remained completely unknown to date. Here, we investigate 11 different FMRFamide-like neuropeptides as putative ligands by surface plasmon resonance with an immobilized recombinant 54 kDa aminoterminal fragment of HC110-R as an interaction partner. AF1, AF10 and PF2 exhibit binding with low affinities as indicated by a K d of 11 μM for AF1, 52 μM for AF10 and 583 μM for PF2. Our data indicate that AF1, AF10, and PF2 are putative natural ligands of HC110-R presumably involved in the control of pharyngeal pumping of nematode worms.

A unique thioredoxin of the parasitic nematode Haemonchus contortus with glutaredoxin activity

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a Km 2.19±1.5 μM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.