Neoblast-like stem cells of Fasciola hepatica.

The common liver fluke (Fasciola hepatica) causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of putative novel targets due to their role in development and homeostasis, including at host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, differentiation, and are sensitive to ionising radiation- all attributes of neoblasts in other flatworms. Changes in cell proliferation were also noted during the early stages of in vitro juvenile growth/development (around four to seven days post excystment), which coincided with a marked reduction in the nuclear area of proliferating cells. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologues associated with neoblast-like cells in Schistosoma mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblast-like cells and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics.

Tissue-specific transcriptome analyses provide new insights into GPCR signalling in adult Schistosoma mansoni.

Schistosomes are blood-dwelling trematodes with global impact on human and animal health. Because medical treatment is currently based on a single drug, praziquantel, there is urgent need for the development of alternative control strategies. The Schistosoma mansoni genome project provides a platform to study and connect the genetic repertoire of schistosomes to specific biological functions essential for successful parasitism. G protein-coupled receptors (GPCRs) form the largest superfamily of transmembrane receptors throughout the Eumetazoan phyla, including platyhelminths. Due to their involvement in diverse biological processes, their pharmacological importance, and proven druggability, GPCRs are promising targets for new anthelmintics. However, to identify candidate receptors, a more detailed understanding of the roles of GPCR signalling in schistosome biology is essential. An updated phylogenetic analysis of the S. mansoni GPCR genome (GPCRome) is presented, facilitated by updated genome data that allowed a more precise annotation of GPCRs. Additionally, we review the current knowledge on GPCR signalling in this parasite and provide new insights into the potential roles of GPCRs in schistosome reproduction based on the findings of a recent tissue-specific transcriptomic study in paired and unpaired S. mansoni. According to the current analysis, GPCRs contribute to gonad-specific functions but also to nongonad, pairing-dependent processes. The latter may regulate gonad-unrelated functions during the multifaceted male-female interaction. Finally, we compare the schistosome GPCRome to that of another parasitic trematode, Fasciola, and discuss the importance of GPCRs to basic and applied research. Phylogenetic analyses display GPCR diversity in free-living and parasitic platyhelminths and suggest diverse functions in schistosomes. Although their roles need to be substantiated by functional studies in the future, the data support the selection of GPCR candidates for basic and applied studies, invigorating the exploitation of this important receptor class for drug discovery against schistosomes but also other trematodes.

Post-genomic progress in helminth parasitology.

Helminth parasitology is an important discipline, which poses often unique technical challenges. One challenge is that helminth parasites, particularly those in humans, are often difficult to obtain alive and in sufficient quantities for study; another is the challenge of studying these organisms in vitro - no helminth parasite life cycle has been fully recapitulated outside of a host. Arguably, the key issue retarding progress in helminth parasitology has been a lack of experimental tools and resources, certainly relative to the riches that have driven many parasitologists to adopt free-living model organisms as surrogate systems. In response to these needs, the past 10-12 years have seen the beginnings of helminth parasitology's journey into the 'omics' era, with the release of abundant sequencing resources, and the functional genomics tools with which to test biological hypotheses. To reflect this progress, the 2019 Autumn Symposium of the British Society for Parasitology was held in Queen's University Belfast on the topic of 'post-genomic progress in helminth parasitology'. This issue presents examples of the current state of play in the field, while this editorial summarizes how genomic datasets and functional genomic tools have stimulated impressive recent progress in our understanding of parasite biology.

Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host.

The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.

Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress.

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB-TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB-TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H(+) antiport.

The genome of the blood fluke Schistosoma mansoni.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Discovery of long non-coding RNAs in the liver fluke, Fasciola hepatica.

Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a custom in silico pipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite, Fasciola hepatica, none of which are conserved with those previously described from Schistosoma mansoni. F. hepatica lncRNAs were distinct from F. hepatica mRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2%) and mRNAs (12.8%) were differentially expressed (p<0.001), supporting a functional role for lncRNAs in F. hepatica life stages. While most lncRNAs (81%) were intergenic, we identified some that overlapped protein coding loci in antisense (13%) or intronic (6%) configurations. We found no unequivocal evidence for correlated developmental expression within positionally correlated lncRNA:mRNA pairs, but global co-expression analysis identified five lncRNA that were inversely co-regulated with 89 mRNAs, including a large number of functionally essential proteases. The presence of micro (mi)RNA binding sites in 3135 lncRNAs indicates the potential for miRNA-based post-transcriptional regulation of lncRNA, and/or their function as competing endogenous (ce)RNAs. The same annotation pipeline identified 24,141 putative lncRNAs in F. gigantica. This first description of lncRNAs in F. hepatica provides an avenue to future functional and comparative genomics studies that will provide a new perspective on a poorly understood aspect of parasite biology.

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica.

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Considering RNAi experimental design in parasitic helminths.

Almost a decade has passed since the first report of RNA interference (RNAi) in a parasitic helminth. Whilst much progress has been made with RNAi informing gene function studies in disparate nematode and flatworm parasites, substantial and seemingly prohibitive difficulties have been encountered in some species, hindering progress. An appraisal of current practices, trends and ideals of RNAi experimental design in parasitic helminths is both timely and necessary for a number of reasons: firstly, the increasing availability of parasitic helminth genome/transcriptome resources means there is a growing need for gene function tools such as RNAi; secondly, fundamental differences and unique challenges exist for parasite species which do not apply to model organisms; thirdly, the inherent variation in experimental design, and reported difficulties with reproducibility undermine confidence. Ideally, RNAi studies of gene function should adopt standardised experimental design to aid reproducibility, interpretation and comparative analyses. Although the huge variations in parasite biology and experimental endpoints make RNAi experimental design standardization difficult or impractical, we must strive to validate RNAi experimentation in helminth parasites. To aid this process we identify multiple approaches to RNAi experimental validation and highlight those which we deem to be critical for gene function studies in helminth parasites.

Developmental Regulation and Functional Prediction of microRNAs in an Expanded Fasciola hepatica miRNome.

The liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analyzed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.

Transcriptomic analysis supports a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles.

Fasciola spp. liver flukes have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, including fhe-let-7a-5p, fhe-mir-124-3p and miRNAs predicted to target Wnt-signalling, which supports a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control.

Pan-phylum In Silico Analyses of Nematode Endocannabinoid Signalling Systems Highlight Novel Opportunities for Parasite Drug Target Discovery.

The endocannabinoid signalling (ECS) system is a complex lipid signalling pathway that modulates diverse physiological processes in both vertebrate and invertebrate systems. In nematodes, knowledge of endocannabinoid (EC) biology is derived primarily from the free-living model species Caenorhabditis elegans, where ECS has been linked to key aspects of nematode biology. The conservation and complexity of nematode ECS beyond C. elegans is largely uncharacterised, undermining the understanding of ECS biology in nematodes including species with key importance to human, veterinary and plant health. In this study we exploited publicly available omics datasets, in silico bioinformatics and phylogenetic analyses to examine the presence, conservation and life stage expression profiles of EC-effectors across phylum Nematoda. Our data demonstrate that: (i) ECS is broadly conserved across phylum Nematoda, including in therapeutically and agriculturally relevant species; (ii) EC-effectors appear to display clade and lifestyle-specific conservation patterns; (iii) filarial species possess a reduced EC-effector complement; (iv) there are key differences between nematode and vertebrate EC-effectors; (v) life stage-, tissue- and sex-specific EC-effector expression profiles suggest a role for ECS in therapeutically relevant parasitic nematodes. To our knowledge, this study represents the most comprehensive characterisation of ECS pathways in phylum Nematoda and inform our understanding of nematode ECS complexity. Fundamental knowledge of nematode ECS systems will seed follow-on functional studies in key nematode parasites to underpin novel drug target discovery efforts.

In silico characterisation of the complete Ly6 protein family in Fasciola gigantica supported through transcriptomics of the newly-excysted juveniles.

Fasciola gigantica is one of the aetiological trematodes associated with fascioliasis, which heavily impacts food-production systems and human and animal welfare on a global scale. In the absence of a vaccine, fascioliasis control and treatment is restricted to pasture management, such as clean grazing, and a limited array of chemotherapies, to which signs of resistance are beginning to appear. Research into novel control strategies is therefore urgently required and the advent of 'omics technologies presents considerable opportunity for novel drug and vaccine target discovery. Here, interrogation of the first available F. gigantica newly excysted juvenile (NEJ) transcriptome revealed several protein families of current interest to parasitic flatworm vaccine research, including orthologues of mammalian complement regulator CD59 of the Ly6 family. Ly6 proteins have previously been identified on the tegument of Schistosoma mansoni and induced protective immunity in vaccination trials. Incorporating the recently available F. gigantica genome, the current work revealed 20 novel Ly6 family members in F. gigantica and, in parallel, significantly extended the F. hepatica complement from 3 to 18 members. Phylogenetic analysis revealed several distinct clades within the family, some of which are unique to Fasciola spp. trematodes. Analysis of available proteomic databases also revealed three of the newly discovered FhLy6s were present in extracellular vesicles, which have previously been prioritised in studying the host-parasite interface. The presentation of this new transcriptomic resource, in addition to the Ly6 family proteins here identified, represents a wealth of opportunity for future vaccine research.

The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea.

BACKGROUND: G protein-coupled receptors (GPCRs) constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum. RESULTS: Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification. CONCLUSIONS: Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

Phylum-Spanning Neuropeptide GPCR Identification and Prioritization: Shaping Drug Target Discovery Pipelines for Nematode Parasite Control.

Nematode parasites undermine human health and global food security. The frontline anthelmintic portfolio used to treat parasitic nematodes is threatened by the escalation of anthelmintic resistance, resulting in a demand for new drug targets for parasite control. Nematode neuropeptide signalling pathways represent an attractive source of novel drug targets which currently remain unexploited. The complexity of the nematode neuropeptidergic system challenges the discovery of new targets for parasite control, however recent advances in parasite 'omics' offers an opportunity for the in silico identification and prioritization of targets to seed anthelmintic discovery pipelines. In this study we employed Hidden Markov Model-based searches to identify ~1059 Caenorhabditis elegans neuropeptide G-protein coupled receptor (Ce-NP-GPCR) encoding gene homologs in the predicted protein datasets of 10 key parasitic nematodes that span several phylogenetic clades and lifestyles. We show that, whilst parasitic nematodes possess a reduced complement of Ce-NP-GPCRs, several receptors are broadly conserved across nematode species. To prioritize the most appealing parasitic nematode NP-GPCR anthelmintic targets, we developed a novel in silico nematode parasite drug target prioritization pipeline that incorporates pan-phylum NP-GPCR conservation, C. elegans-derived reverse genetics phenotype, and parasite life-stage specific expression datasets. Several NP-GPCRs emerge as the most attractive anthelmintic targets for broad spectrum nematode parasite control. Our analyses have also identified the most appropriate targets for species- and life stage- directed chemotherapies; in this context we have identified several NP-GPCRs with macrofilaricidal potential. These data focus functional validation efforts towards the most appealing NP-GPCR targets and, in addition, the prioritization strategy employed here provides a blueprint for parasitic nematode target selection beyond NP-GPCRs.

Uptake of macromolecules by cercariae during skin penetration and transformation to schistosomula (Schistosoma mansoni).

Here, we observed the uptake of membrane-impermeant molecules by cercariae as they penetrate the skin and are transformed into schistosomula. We propose that membrane-impermeant molecules, Lucifer Yellow, Propidium iodide and Hoechst 33258 enter the parasite through both thenephridiopore and the surface membrane and then diffuse throughout the body of the parasite. We present a hypothesis that the internal cells of the body of the schistosomulum represent a new host-parasite interface, at which skin-derived growth factors may stimulate receptors on internal membranes during transformation of the cercariae into the schistosomulum.

Molecular characterisation and vaccine efficacy of two novel developmentally regulated surface tegument proteins of Fasciola hepatica.

The surface tegument of Fasciola hepatica is a crucial tissue due to its key role at the host-parasite interface. We characterised three novel proteins, termed Fhteg1, Fhteg5 and Fhteg8, that are found in the tegument membrane fraction of adult F. hepatica. Bioinformatic analysis of proteomic datasets identified Fhteg5 and Fhteg8 as tegumental glycoproteins and revealed that Fhteg1, Fhteg5 and Fhteg8 are associated with exosomes of adult F. hepatica. Fhteg1, Fhteg5 and Fhteg8 appear to be related to uncharacterised sequences in F. gigantica, Fasciolopsis buski, Echinostoma caproni, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum and S. mansoni, although F. hepatica appears to have expanded this family. Fhteg1 and Fhteg5 were characterised in detail. The Fhteg1 and Fhteg5 gene transcripts each demonstrate significant upregulation in juvenile fluke 2-4 days post-excystment, with transcript levels maintained during development over 3 weeks in vitro. RNAseq data showed that both Fhtegs are expressed in the adult life stage, although the transcript levels were about 8 fold lower than those in juveniles (3 week post infection). Using immunocytochemistry, Fhteg1 and Fhteg5 were each shown to be expressed in cells adjacent to the muscle layer as well as on the surface of 1 week old juveniles, whilst Fhteg5 was also present in cells at the base of the pharynx. RNAi mediated knockdown of Fhteg1 and Fhteg5 transcripts in 4-10 day old juveniles had no effect on parasite survival, movement or growth in vitro. Although no IgG responses were observed for Fhteg1 or Fhteg5 during infection in sheep and cattle, both proteins elicited a low IgG response in a proportion of infected rats. Rats vaccinated with Fhteg1 and Fhteg5 showed good IgG responses to both proteins and a mean 48.2 % reduction in worm burden following parasite challenge. Although vaccination of cattle with both proteins induced a range of IgG responses, no protection was observed against parasite challenge. This is the first study to provide insights into the molecular properties of two novel, developmentally regulated surface tegument proteins in F. hepatica.