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[Display omitted] • Entomopathogenic nematodes are excellent models for studying immunomodulation. • Entomopathogenic nematodes secrete virulence factors that promote infection. • This information is critical for advancing the biocontrol of insect pests. Much of the available knowledge of entomopathogenic virulence factors has been gleaned from studies in the nematode parasite Steinernema carpocapsae , but there is good reason to complement this knowledge with similar studies in Heterorhabditis bacteriophora. Three candidate virulence factors from H. bacteriophora have recently been characterised, and each was demonstrated to contribute to infection. This information can be used not only to advance efforts in the biocontrol of insect pests, but also to make inferences about the emergence of parasitism among Clade V nematodes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Here we report on NemChR-DB, an integrative database of parasitic nematode chemoreceptors. • NemChR-DB contains sequence, structural and literature-based annotations for performing cross-species comparisons. • NemChR-DB currently houses chemoreceptor sequence data from 53 nematode species representing 28 unique families. Nematode Chemosensory G-Protein Coupled Receptors have expanded within nematodes, where they play important roles in foraging and host-seeking behaviour. Nematode Chemosensory G-Protein Coupled Receptors are most highly expressed during free-living stages when chemosensory signalling is required for host detection and nematode activation in various parasitic nematodes, and therefore position Nematode Chemosensory G-Protein Coupled Receptors at the transition from infective to parasitic stages, making them important regulators to study in terms of host-seeking and host specificity. To facilitate the analysis of Nematode Chemosensory G-Protein Coupled Receptors, here we describe an integrative database of nematode chemoreceptors called NemChR-DB. This database enables users to study diverse parasitic nematode chemoreceptors, functionally explore sequence entries through structural and literature-based annotations, and perform cross-species comparisons. [ABSTRACT FROM AUTHOR]

The screening of candidate compounds and natural products for anthelmintic activity is important for discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device (‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm's pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity that is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae in the microfluidic EPG platform. Ancylostoma ceylanicum and A. caninum infective L3s (iL3s) that had been activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps as seen in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (termed a ‘flutter’); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG ‘events.’ EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lungs exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior. [ABSTRACT FROM AUTHOR]

Soil-transmitted helminthiases (STHs) collectively cause the highest global burden of parasitic disease after malaria and are most prevalent in the poorest communities, especially in sub-Saharan Africa. Climate change is predicted to alter the physical environment through cumulative impacts of warming and extreme fluctuations in temperature and precipitation, with cascading effects on human health and wellbeing, food security and socioeconomic infrastructure. Understanding how the spectrum of climate change effects will influence STHs is therefore of critical importance to the control of the global burden of human parasitic disease. Realistic progress in the global control of STH in a changing climate requires a multidisciplinary approach that includes the sciences (e.g. thermal thresholds for parasite development and resilience) and social sciences (e.g. behavior and implementation of education and sanitation programs). [Copyright &y& Elsevier]

The developmentally arrested hookworm infective larva resumes development only after encountering specific host-mediated cues during invasion. These cues activate a signaling pathway that culminates in the resumption of development. In Ancylostoma caninum, activation is characterised by the resumption of feeding and the release of excretory/secretory products required for infection. The dauer stage of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm infective larva. Dauer larvae exit developmental arrest in response to environmental cues that indicate favorable conditions for reproduction and growth. Because of the similarity between dauer recovery and activation, exit from dauer provides a model for hookworm larval activation. An insulin-signaling pathway has been implicated in controlling exit from developmental arrest in both C. elegans dauers and A. caninum larvae. To further investigate the role of insulin signaling in hookworm larval activation, the phosphatidylinositol-3-OH kinase inhibitor LY294002 was tested for its effect on in vitro activation using the resumption of feeding as a marker for activation. LY294002 prevented feeding in A. caninum infective larvae stimulated with host serum filtrate and a glutathione-analogue, the muscarinic agonist arecoline, or the cell permeable cGMP-analogue 8-bromo-cGMP. Similar results were seen with the congeneric hookworm Ancylostoma ceylanicum. These data suggest that an insulin-signaling pathway mediates activation in hookworm larvae, as in C. elegans, and that the phosphatidylinositol-3-OH kinase inhibitor acts downstream of the cGMP and muscarinic signaling steps in the pathway. In A. caninum, LY294002 had no effect on the release of excretory/secretory products associated with activation, suggesting that the secretory pathway diverges from the activation pathway upstream of the phosphatidylinositol-3-OH kinase step. These results provide additional support for the insulin-signaling pathway as the primarily pathway for activation to parasitism in hookworm larvae. [Copyright &y& Elsevier]

The developmentally arrested infective larva (L3) of hookworms encounters a host-specific signal during infection that initiates previously suspended developmental pathways. Activated L3 express a parasitic gene set that encodes proteins involved in moulting, growth and development to the adult stage. Early events in this activation to parasitism can be investigated using an in vitro larval feeding assay. When Ancylostoma caninum L3 are exposed to a host-like stimulus, they resume feeding and release molecules involved in infection. The dauer larva of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm L3. Recovery from the dauer stage has been proposed as a model for the transition to parasitism in hookworm. Dauer formation and recovery involve several tightly regulated pathways, including a cyclic GMP mediated signalling pathway. To determine if hookworm L3 activation uses a similar pathway, 8-bromo-cyclic GMP, a membrane permeant analogue of cyclic GMP, was tested for its ability to stimulate feeding. Populations of L3 incubated with 0.5 mM 8-bromo-cyclic GMP began feeding, and reached maximum feeding at 3.5–5.0 mM. Unlike the serum stimulus, which triggers feeding after a short exposure, 8-bromo-cyclic GMP must be present throughout the entire incubation. Both serum stimulated and 8-bromo-cyclic GMP stimulated L3 secreted Ancylostoma secreted protein 1, indicating that the stimuli activate the same pathway. Serum and 8-bromo-cyclic GMP stimulated feeding was inhibited by atropine, a muscarinic receptor antagonist. However, only serum stimulated feeding was inhibited by 4,7-phenanthroline, a non-chelating isomer of the metalloprotease inhibitor 1,10-phenantholine. The results indicate that cyclic GMP mediates activation in hookworm larvae, and that a muscarinic receptor is involved in activation. This also suggests that hookworm activation and dauer recovery share similar signalling pathways, and that C. elegans dauer recovery can be used as a model for the transition to parasitism in hookworms. [Copyright &y& Elsevier]

Abstract: When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five–sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two–threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection. [Copyright &y& Elsevier]

Abstract: Infective hookworm L3s encounter a host-specific signal during infection that re-initiates a suspended developmental pathway, resulting in development to the adult stage. This resumption of development in the host is analogous to recovery of developmentally arrested Caenorhabditis elegans dauer larvae in response to favorable environmental signals. Dauer recovery in C. elegans dauers and hookworm L3s is mediated by insulin-like signaling (ILS). A key output of ILS in C. elegans is the forkhead transcription factor DAF-16, which controls the expression of genes required for maintenance of the dauer stage. The similarity between recovery pathways of L3s and dauers suggests that DAF-16 functions similarly in hookworm L3 activation. To test this, orthologs of Ce-DAF-16 were isolated from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. The protein sequences of hookworm DAF-16 DNA binding domains were identical, and shared 94% identity with the b and c isoforms of Ce-DAF-16. Ac-DAF-16 expressed in HEK293 kidney cells bound strongly to the conserved DAF family binding element (DBE), but not to a random DNA sequence. Ac-DAF-16 was able to drive transcription of a reporter gene located downstream of six copies of the DBE in NIH3T3 cells under starved conditions. Addition of serum caused a decrease in reporter gene expression, indicating that DAF-16 is negatively regulated by growth factor stimulation. These data confirm the presence of DAF-16 orthologs in hookworms, and demonstrate that Ac-DAF-16 binds to and drives transcription from a conserved DAF-16 family DNA binding element. [Copyright &y& Elsevier]

Nematode virulence factors are of interest for a variety of applications including biocontrol against insect pests and the alleviation of autoimmune diseases with nematode-derived factors. In silico "omics" techniques have generated a wealth of candidate factors that may be important in the establishment of nematode infections, although the challenge of characterizing these individual factors in vivo remains. Here we provide a fundamental characterization of a putative lysozyme and serine carboxypeptidase from the host-induced transcriptome of Heterorhabditis bacteriophora. Both factors accelerated the mortality rate following Drosophila melanogaster infections with Photorhabdus luminescens , and both factors suppressed phenoloxidase activity in D. melanogaster hemolymph. Furthermore, the serine carboxypeptidase was lethal to a subpopulation of flies and suppressed the upregulation of antimicrobial peptides as well as phagocytosis. Together, our findings suggest that this serine carboxypeptidase possess both toxic and immunomodulatory properties while the lysozyme is likely to confer immunomodulatory, but not toxic effects. Image 1 • Heterorhabditis upregulates a serine carboxypeptidase and lysozyme in response to host factors. • Recombinant versions accelerate killing during a Photorhabdus infection in Drosophila. • The serine carboxypeptidase limits phenoloxidase activity, AMP upregulation, and phagocytosis. • The lysozyme increases Photorhabdus bacterial load and limits phenoloxidase activity. [ABSTRACT FROM AUTHOR]

• The Ancylostoma ceylanicum genome encodes 371 G-coupled protein receptors. • Seventy-four receptors are differentially expressed in L4s. • Thirty-one of the differentially expressed receptors are nematode chemoreceptors. • Chemosensory receptor diversity decreases by nearly half at the fourth stage moult. • Two chemoreceptor promoters were expressed in transgenic Caenorhabditis elegans. Free-living nematodes respond to variable and unpredictable environmental stimuli whereas parasitic nematodes exist in a more stable host environment. A positive correlation between the presence of environmental stages in the nematode life cycle and an increasing number of G-protein coupled receptors (GPCRs) reflects this difference in free-living and parasitic lifestyles. As hookworm larvae move from the external environment into a host, they detect uncharacterized host components, initiating a signalling cascade that results in the resumption of development and eventual maturation. Previous studies suggest this process is mediated by GPCRs in amphidial neurons. Here we set out to uncover candidate GPCRs required by a hookworm to recognise its host. First, we identified all potential Ancylostoma ceylanicum GPCRs encoded in the genome. We then used life cycle stage-specific RNA-seq data to identify differentially expressed GPCRs between the free-living infective L3 (iL3) and subsequent parasitic stages to identify receptors involved in the transition to parasitism. We reasoned that GPCRs involved in host recognition and developmental activation would be expressed at higher levels in the environmental iL3 stage than in subsequent stages. Our results support the model that a decrease in GPCR diversity occurs as the larvae develop from the free-living iL3 stage to the parasitic L3 (pL3) in the host over 24–72 h. We find that overall GPCR expression and diversity is highest in the iL3 compared with subsequent parasitic stages. By 72 h, there was an approximately 50% decrease in GPCR richness associated with the moult from the pL3 to the L4. Taken together, our data uncover a negative correlation between GPCR diversity and parasitic development in hookworm. Finally, we demonstrate proof of principal that Caenorhabditis elegans can be used as a heterologous system to examine the expression pattern of candidate host signal chemoreceptors (CRs) from hookworm. We observe expression of candidate host signal CRs in C. elegans , demonstrating that C. elegans can be effectively used as a surrogate to identify expressed hookworm genes. We present several preliminary examples of this strategy and confirm a candidate CR as neuronally expressed. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Ancylostoma caninum strain KGR is phenotypically resistant to thiabendazole. • Aca -KGR has a fixed, non-synonymous single nucleotide polymorphism in β-tubulin isotype 1 codon 167. • CRISPR/Cas9 editing of the orthologous Caenorhabditis elegans gene confers resistance. • Aca -KGR is also phenotypically resistant to ivermectin. Abstract Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment. [ABSTRACT FROM AUTHOR]

Interest has recently grown in developing the entomopathogenic nematode Heterorhabditis bacteriophora as a model to genetically dissect the process of parasitic infection. Despite the availability of a full genome assembly, there is substantial variation in gene model accuracy. Here, a methodology is presented for leveraging RNA-seq evidence to generate improved annotations using ab initio gene prediction software. After alignment of reads and subsequent generation of a RNA-seq supported annotation, the new gene prediction models were verified on a selection of genes by comparison with sequenced 5′ and 3′ rapid amplification of cDNA ends products. By utilising a whole transcriptome for genome annotation, the current reference annotation was enriched, demonstrating the importance of coupling transcriptional data with genome assemblies. [ABSTRACT FROM AUTHOR]

Abstract: Infective hookworm L3 encounter a host specific signal during invasion that re-activates suspended developmental pathways. Response to this cue is critical for the successful infection and completion of the life cycle in the host. In the free-living nematode Caenorhabditis elegans, recovery from the developmentally arrested dauer stage in response to environmental cues is analogous to the resumption of development in invading hookworm L3. Transforming growth factor β (TGF-β) and insulin-like signalling pathways mediate dauer formation and recovery. An insulin-like signalling pathway mediates L3 activation in hookworms. To determine the role of TGF-β signalling in hookworm infection, an ortholog of the C. elegans TGF-β signalling molecule daf-7 was cloned and characterised. Sequence from a hookworm expressed sequence tag was used to design specific primers for PCR amplification of Ac-daf-7 from Ancylostoma caninum infective L3 cDNA. Amplicons from the 5′ and 3′ ends were cloned, sequenced, and combined to create a full-length composite Ac-daf-7 cDNA sequence. The 1634 nucleotide cDNA encoded a 355 amino acid open reading frame with significant homology to Ce-DAF-7 and other TGF-β signalling molecules. The deduced amino acid sequence contained seven conserved cysteines characteristic of TGF-β family members, as well as two additional conserved cysteines found in members of the TGF-β/activin subfamily. Ac-DAF-7 contains a characteristic C-terminal ligand domain that is predicted to be released from a propeptide by proteolytic cleavage at a tetrabasic cleavage site. Ac-daf-7 mRNA was strongly detected by reverse transcriptase PCR in L3 and serum stimulated L3 cDNA, and weakly in cDNA from L1 and adult life cycle stages. Antiserum against Escherichia coli expressed recombinant Ac-DAF-7 detected the mature protein in L3 and adult soluble extracts, but not in excretory/secretory products from serum stimulated L3 or adults. Increased expression in arrested L3 stages suggests that Ac-daf-7 is important for developmental arrest. [Copyright &y& Elsevier]

Abstract: Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5′ and 3′ ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme–inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine. [Copyright &y& Elsevier]

A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L3) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L3. Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L3 and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L3 into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen. [Copyright &y& Elsevier]

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics. [Copyright &y& Elsevier]

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3–6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship. [Copyright &y& Elsevier]

[Display omitted] • Describe a chemogenomic pipeline for the identification of anthelmintics that target nematode chemoreceptor GPCRs. • Identify differentially expressed GPCRs in Heterorhabditis bacteriophora after host-activation. • Characterize candidate anthelmintics and substrates in Heterorhabditis bacteriophora. Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates. [ABSTRACT FROM AUTHOR]