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Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Schistosoma haematobiumis the most prevalent of the human-infecting schistosome species, causing significant morbidity in endemically exposed populations. Despite this, it has been relatively understudied compared to its fellow species,S. mansoni. Here we provide the first comprehensive characterization of theS. haematobiumTegument Allergen-Like protein family, a key protein family directly linked to protective immunity inS. mansoniinfection. Comparable with observations forS. mansoni, parasite phylogenetic analysis and relative gene expression combined with host serological analysis support a cross-reactive relationship betweenS. haematobiumTAL proteins, exposed to the host immune system as adult worms die, and closely related proteins, exposed during penetration by the infecting cercarial and early schistosomulae stages. Specifically, our results strengthen the evidence for host immunity driven by cross-reactivity between family members TAL3 and TAL5, establishing it for the first time forS. haematobiuminfection. Furthermore, we build upon this relationship to include the involvement of an additional member of the TAL protein family, TAL11 for both schistosome species. Finally, we show a close association between experience of infection and intensity of transmission and the development of protective IgE responses to these antigens, thus improving our knowledge of the mechanisms by which protective host immune responses develop. This knowledge will be critical in understanding how control efforts such as mass drug administration campaigns influence the development of host immunity and subsequent patterns of infection and disease within endemic populations.Author SummaryS. haematobiumis the most prevalent of the human infecting schistosomes. Along withS. mansoni, it is responsible for the majority of schistosomiasis cases that are borne by the populations of sub-Saharan Africa, where the global burden of this infection is centered. Here, we provide insight into the IgE antibody response that protects against these infections. Through utilization ofin silicoanalysis and transcriptional studies of parasite life stages, in combination with immuno-epidemiological studies, we explore the relationship between host immune protection and a parasite protein family named the Tegument Allergen-Like (TAL) proteins. Our results show that several members of the TAL protein family are important in host protection to both these major schistosome species. For the first time we demonstrate that a progressive cross-reactive TAL-IgE response occurs againstS. haematobium, similar to that previous observed inS. mansoniinfection. We additionally expand upon previous knowledge forS. mansoni, identifying further complexity in the cross-reactive relationship between TAL family members, providing evidence of a key role for family member TAL11 in induction of the protective host immune response.

Background: Schistosoma mansoni, a parasitic worm species responsible for the neglected tropical disease schistosomiasis, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic and epigenetic processes. As inhibition of S. mansoni epigenetic machinery components impairs key transitions throughout the parasite’s digenetic lifecycle, a greater understanding of how epi-drugs affect molecular processes in schistosomes could lead to the development of new anthelmintics. Methods: In vitro whole organism assays were used to assess the anti-schistosomal activity of 39 Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors on different parasite life cycle stages. Moreover, tissue-specific stains and genomic analysis shed light on the effect of these small molecules on the parasite biology. Results: Amongst this collection of small molecules, compound 33 was the most potent in reducing ex vivo viabilities of schistosomula, juveniles, miracidia and adults. At its sub-lethal concentration to adults (3.13 µM), compound 33 also significantly impacted oviposition, ovarian as well as vitellarian architecture and gonadal/neoblast stem cell proliferation. ATAC-seq analysis of adults demonstrated that compound 33 significantly affected chromatin structure (intragenic regions > intergenic regions), especially in genes differentially expressed in cell populations (e.g., germinal stem cells, hes2+ stem cell progeny, S1 cells and late female germinal cells) associated with these ex vivo phenotypes. KEGG analyses further highlighted that chromatin structure of genes associated with sugar metabolism as well as TGF-beta and Wnt signalling were also significantly perturbed by compound 33 treatment. Conclusions: This work confirms the importance of histone methylation in S. mansoni lifecycle transitions, suggesting that evaluation of LSD1 - targeting epi-drugs may facilitate the search for next-generation anti-schistosomal drugs. The ability of compound 33 to modulate chromatin structure as well as inhibit parasite survival, oviposition and stem cell proliferation warrants further investigations of this compound and its epigenetic target SmLSD1.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p, Author summary Schistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni α-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.

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.

Funder: IBERS, Aberystwyth University PhD studentship, Funder: Higher Education Funding Council for Wales (HEFCW) - Global Challenges Research Fund (GCRF), Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki-diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, psiLuc treated females, psmp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Pharmacological inhibition of SmNAGAL may lead to the development of a novel anthelmintic class of compounds.Author summarySchistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni α-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 μM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 μM, 0.20 μM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies., Graphical abstract Image 1, Highlights • Lead compound 22 was identified with EC50 of 0.44 µM and 84.7 nM for schistosomula and adult worms. • 20 analogues of the lead compound 22 were synthesised. • Compounds 25, 30 and 32 showed the best selectivity profile. • Compounds 31 and 33 are the most active on three medically important schistosome species.

Schistosomiasis is a neglected tropical disease that affects more than 250 million people worldwide. The only drug available for its treatment undergoes first-pass hepatic metabolism and is not capable of preventing reinfection, which makes the search of new therapies urgently needed. Due to the essential role of fumarases in metabolism, these enzymes represent potential targets for developing novel schistosomiasis treatments. Here, we evaluate the expression profiles for class I and class II fumarases from Schistosoma mansoni (SmFHI and SmFHII, respectively), and report the complete characterization of SmFHII. The first SmFHII structure in complex with L-malate was determined at 1.85 A resolution. The significant thermoshift observed for SmFHII in the presence of identified ligands makes the differential scanning fluorimetry an adequate technique for ligand screening. A complete kinetic characterization of SmFHII was performed, and comparison with the human fumarase (HsFH) revealed differences regarding the turnover number (kcat). Structural characterization allowed us to identify differences between SmFHII and HsFH that could be explored to design new selective inhibitors. This work represents the very first step towards validate the fumarases as drug targets to treat schistosomiasis. Our results provide the structural basis to rational search for selective ligands.

Background: Schistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease of considerable importance in resource-poor communities throughout the developing world. In the absence of an immunoprophylactic vaccine and due to over-reliance on a single chemotherapy (praziquantel), schistosomiasis control is at risk should drug insensitive schistosomes develop. In this context, application of in silico virtual screening on validated schistosome targets has proven successful in the identification of novel small molecules with anti-schistosomal activity. Methods: Focusing on the Schistosoma mansoni histone methylation machinery, we herein have used RNA interference (RNAi), ELISA-mediated detection of H3K4 methylation, homology modelling and in silico virtual screening to identify a small collection of small molecules for anti-schistosomal testing. A combination of low to high-throughput whole organism assays were subsequently used to assess these compounds’ activities on miracidia to sporocyst transformation, schistosomula phenotype/motility metrics and adult worm motility/oviposition readouts. Results: RNAi-mediated knockdown of smp_138030/smmll-1 (encoding a histone methyltransferase, HMT) in adult worms (~60%) reduced parasite motility and egg production. Moreover, in silico docking of compounds into Smp_138030/SmMLL-1’s homology model highlighted competitive substrate pocket inhibitors, some of which demonstrated significant activity on miracidia, schistosomula and adult worm lifecycle stages together with variable effects on HepG2 cells. Particularly, the effect of compounds containing a 6-(piperazin-1-yl)-1,3,5-triazine core on adult schistosomes recapitulated the results of the smp_138030/smmll-1 RNAi screens. Conclusions: The biological data and the structure-activity relationship presented in this study define the 6-(piperazin-1-yl)-1,3,5-triazine core as a promising starting point in ongoing efforts to develop new urgently needed schistosomicides.

Schistosomiasis is a chronically-debilitating neglected tropical disease (NTD) that predominantly affects people living in resource-poor communities of tropical and subtropical countries. Schistosoma mansoni, one of three species responsible for most human infections, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic- and epigenetic- processes. As inhibition of S. mansoni epigenetic machinery components has been shown to impair key transitions throughout the parasite’s digenetic lifecycle, this knowledge is currently fuelling the search for new epi-drug - based anthelmintics.In this study, the anti-schistosomal activity of 39 re-purposed Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors was investigated on key life cycle stages associated with both definitive (schistosomula, juvenile worms, sexually-mature adults) and intermediate host (miracidia) infection. The most active compound (compound 33; e.g. schistosomula phenotype EC50 = 4.370 µM; adult worm motility EC50 = 2.137 µM) was subsequently used to provide further insight into the critical role of S. mansoni lysine specific demethylase 1 (SmLSD1) in adult worm oviposition and stem cell proliferation. Here, compound 33 treatment of adult schistosomes led to significant defects in egg production, intra-egg vitellocyte/ovum packaging and gonadal/neoblast stem cell proliferation. A greater abundance of H3K4me2 marks accompanied these phenotypes and supported specific inhibition of SmLSD1 in adult schistosomes by compound 33. In silico screening indicated that compound 33 likely inhibits SmLSD1 activity by covalently reacting with the FAD cofactor.This work suggests that evaluation of HsLSD1 - targeting epi-drugs could have utility in the search for next-generation anti-schistosomals. The ability of compound 33 to inhibit parasite survival, oviposition, H3K4me2 demethylation and stem cell proliferation warrants further investigations of this compound and its epigenetic target. This data further highlights the importance of histone methylation in S. mansoni lifecycle transitions.Author summaryAffecting over 200 million people, schistosomiasis is a chronic disease caused by the parasitic worm Schistosoma mansoni. The frontline drug for schistosomiasis treatment is praziquantel. Owing to the concern surrounding praziquantel insensitivity or resistance developing, current research is directed towards the identification of novel drugs. We have focused our search for compounds that affect essential aspects of schistosome biology including parasite movement, fertility, cell proliferation and survival. Since all of these functions are potentially influenced by epigenetic regulation of gene expression, we investigated the activity of compounds that alter histone methylation status. In this report, we show that S. mansoni Lysine Specific Demethylase 1 (SmLSD1), a histone demethylase, is critical to miracidia-to-sporocyst transitioning, adult worm motility, egg production and parasite survival. Inhibition of SmLSD1 with compounds developed to inhibit the human paralog show promising potential as novel anti-schistosomal agents.

Graphical abstract, Highlights • Smval6 is expressed in oral/ventral suckers, oesophageal gland and mesenchymal cells of Schistosoma mansoni. • Smval6 knockdown increases surface membrane permeability. • SmVAL6 interacts with Sm14 and DLC proteins., The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

Schistosomiasis endangers the lives of greater than 200 million people every year and is predominantly controlled by a single class chemotherapy, praziquantel (PZQ). Development of PZQ replacement (to combat the threat of PZQ insensitivity/resistance arising) or combinatorial (to facilitate the killing of PZQ-insensitive juvenile schistosomes) chemotherapies would help sustain this control strategy into the future. Here, we re-categorise two families of druggable epigenetic targets in Schistosoma mansoni, the histone methyltransferases (HMTs) and the histone demethylases (HDMs). Amongst these, a S. mansoni Lysine Specific Demethylase 1 (SmLSD1, Smp_150560) homolog was selected for further analyses. Homology modelling of SmLSD1 and in silico docking of greater than four thousand putative inhibitors identified seven (L1 – L7) showing more favourable binding to the target pocket of SmLSD1 vs Homo sapiens HsLSD1; six of these seven (L1 – L6) plus three structural analogues of L7 (L8 – L10) were subsequently screened against schistosomula using the Roboworm anthelmintic discovery platform. The most active compounds (L10 - pirarubicin > L8 – danunorubicin hydrochloride) were subsequently tested against juvenile (3 wk old) and mature (7 wk old) schistosome stages and found to impede motility, suppress egg production and affect tegumental surfaces. When compared to a surrogate human cell line (HepG2), a moderate window of selectivity was observed for the most active compound L10 (selectivity indices - 11 for schistosomula, 9 for juveniles, 1.5 for adults). Finally, RNA interference of SmLSD1 recapitulated the egg-laying defect of schistosomes co-cultivated in the presence of L10 and L8. These preliminary results suggest that SmLSD1 represents an attractive new target for schistosomiasis; identification of more potent and selective SmLSD1 compounds, however, is essential. Nevertheless, the approaches described herein highlight an interdisciplinary strategy for selecting and screening novel/repositioned anti-schistosomals, which can be applied to any druggable (epigenetic) target encoded by the parasite's genome., Graphical abstract Image 1, Highlights • Schistosoma mansoni contains 27 histone methyltransferases (HMTs) and 14 histone demethylases (HDMs). • S. mansoni lysine specific demethylase 1 (SmLSD1) is a druggable target. • Schistosomes treated with the putative SmLSD1 inhibitor pirarubicin or siRNAs targeting SmLSD1 are less fecund.

Background Praziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium. Methodology/Principle findings Thirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 μM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor). Conclusion/Significance Collectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control., Author summary Human schistosomiasis is caused by infection with parasitic blood fluke worms. Global control of this NTD is currently facilitated by administration of a single drug, praziquantel (PZQ). This mono-chemotherapeutic strategy of schistosomiasis control presents challenges as PZQ is not active against all human-dwelling schistosome lifecycle stages and the evolution of PZQ resistant parasites remains a threat. Therefore, new drugs to be used in combination with or in replacement of PZQ are urgently needed. Here, continuing our studies on Schistosoma mansoni epigenetic processes, we performed anthelmintic screening of 37 epigenetic probes/epigenetic inhibitors obtained from the Structural Genomics Consortium (SGC). The results of these studies highlighted that schistosome protein methylation/demethylation processes are acutely vulnerable. In particular, compounds affecting schistosome SMYD (LLY-507, BAY-598) or JMJD (GSK-J4) homologues are especially active on schistosomula and adult worms during in vitro phenotypic drug screens. The active epigenetic probes identified here as well as their corresponding S. mansoni protein targets offers new starting points for the development of next-generation anti-schistosomals.

Graphical abstract, Highlights • The Schistosoma mansoni venom allergen-like (SmVAL) family relationships were investigated. • Transcription patterns of SmVALs associate with phylogenetic relationships. • There was clear antibody cross-reactivity between related native SmVAL proteins. • SmVAL4, 10, 18 and 19 all localized via WISH to pre-acetabular glands of cercariae., Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A–D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity.

Summary While antigens from Schistosoma schistosomula have been suggested as potential vaccine candidates, the association between antibody responses with schistosomula antigens and infection intensity at reinfection is not well known. Schistosoma mansoni‐infected individuals were recruited from a schistosomiasis endemic area in Uganda (n = 372), treated with 40 mg/kg praziquantel (PZQ) and followed up at five weeks and at one year post‐treatment. Pre‐treatment and five weeks post‐treatment immunoglobulin (Ig) E, IgG1 and IgG4 levels against recombinant schistosomula antigens rSmKK7, rSmLy6A, rSmLy6B and rSmTSP7 were measured using ELISA. Factors associated with detectable pre‐treatment or post‐treatment antibody response against the schistosomula antigens and the association between five‐week antibody responses and one year post‐treatment reinfection intensity among antibody responders were examined. Being male was associated with higher pre‐treatment IgG1 to rSmKK7, rSmLy6a and AWA. Five weeks post‐treatment antibody responses against schistosomula antigens were not associated with one year post‐treatment reinfection intensity among antibody responders’ antibody levels against rSmKK7, rSmLy6B and rSmTSP7 dropped, but increased against rSmLy6A, AWA and SEA at five weeks post‐treatment among antibody responders. S. mansoni‐infected individuals exhibit detectable antibody responses to schistosomula antigens that are affected by treatment. These findings indicate that schistosomula antigens induce highly varied antibody responses and could have implications for vaccine development.

Well-informed choices about how to improve health outcomes depend on access to reliable information, including research evidence. Many people (the public, health professionals and policymakers) have problems understanding some of the terminology used in describing evaluations of treatments. In this article, we describe the development of a glossary to provide plain language explanations of commonly used terms. We identified terms for inclusion in the glossary by screening multiple sources, including 14 other glossaries. Using an iterative process that included feedback and user testing, we designed an online glossary that is freely available at http://getitglossary.org. The glossary includes 242 commonly used terms. For each entry there is a suggested preferred term (plain language term), a short definition that can be accessed as a 'pop-up' within text, synonyms, a longer explanation and links to related terms. We also flag 11 terms that we advise against using because they are ambiguous or misinterpreted, with explanations of why we advise against using them. The glossary can be incorporated into documents, providing support for evidence-informed health-care decisions, and embedded in other websites serving a wide variety of functions.

Schistosomiasis endangers the lives of greater than 200 million people every year and is predominantly controlled by a single class chemotherapy, praziquantel (PZQ). Development of PZQ replacement (to combat the threat of PZQ insensitivity/resistance arising) or combinatorial (to facilitate the killing of PZQ-insensitive juvenile schistosomes) chemotherapies would help sustain this control strategy into the future. Here, we re-categorise two families of druggable epigenetic targets in Schistosoma mansoni, the histone methyltransferases (HMTs) and the histone demethylases (HDMs). Amongst these, a S. mansoni Lysine Specific Demethylase 1 (SmLSD1, Smp_150560) homolog was selected for further analyses. Homology modelling of SmLSD1 and in silico docking of greater than four thousand putative inhibitors identified seven (L1 - L7) showing more favourable binding to the target pocket of SmLSD1 vs Homo sapiens HsLSD1; six of these seven (L1 - L6) plus three structural analogues of L7 (L8 - L10) were subsequently screened against schistosomula using the Roboworm anthelmintic discovery platform. The most active compounds (L10 - pirarubicin L8 - danunorubicin hydrochloride) were subsequently tested against juvenile (3 wk old) and mature (7 wk old) schistosome stages and found to impede motility, suppress egg production and affect tegumental surfaces. When compared to a surrogate human cell line (HepG2), a moderate window of selectivity was observed for the most active compound L10 (selectivity indices - 11 for schistosomula, 9 for juveniles, 1.5 for adults). Finally, RNA interference of SmLSD1 recapitulated the egg-laying defect of schistosomes co-cultivated in the presence of L10 and L8. These preliminary results suggest that SmLSD1 represents an attractive new target for schistosomiasis; identification of more potent and selective SmLSD1 compounds, however, is essential. Nevertheless, the approaches described herein highlight an interdisciplinary strategy for selecting and screening novel/repositioned anti-schistosomals, which can be applied to any druggable (epigenetic) target encoded by the parasite's genome.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

Background The debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail’s response to infection. Methodology/Principle findings Here, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail’s DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species’ genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP). Conclusions/Significance The presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategies., Author summary Members of the genus Biomphalaria represent air-breathing (pulmonate) aquatic gastropod molluscs of great medical importance. The majority of species are obligatory intermediate hosts of the trematode flatworm Schistosoma mansoni, a pathogen responsible for the devastating neglected tropical disease schistosomiasis. Since the spread of the disease is governed by the temperature tolerance of its molluscan host, the envisaged rise in global temperatures will allow for the further expansion of the snail outside the native range into temperate regions. While schistosomiasis is currently predominantly controlled by praziquantel-mediated chemotherapy of infected individuals, novel strategies are needed in the longer term. The availability of the new Biomphalaria glabrata genome importantly now enables the design of next-generation schistosomiasis control strategies focused on the intermediate host. Here, using a variety of diverse approaches, we functionally characterise the critically important molecular process DNA methylation in B. glabrata, which is mediated by a suite of biological participants and is involved in a wide range of metazoan functions. Importantly, we confirm the presence of 5mC within the snail’s genome using global as well as locus-specific methodologies and we further provide the first evidence for a S. mansoni-provoked modulation of the intermediate’s host DNA methylation system.

SUMMARYDuring platyhelminth infection, a cocktail of proteins is released by the parasite to aid invasion, initiate feeding, facilitate adaptation and mediate modulation of the host immune response. Included amongst these proteins is the Venom Allergen-Like (VAL) family, part of the largerspermcoatingprotein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) superfamily. To explore the significance of this protein family during Platyhelminthes development and host interactions, we systematically summarize all published proteomic, genomic and immunological investigations of the VAL protein family to date. By conducting new genomic and transcriptomic interrogations to identify over 200 VAL proteins (228) from species in all 4 traditional taxonomic classes (Trematoda, Cestoda, Monogenea and Turbellaria), we further expand our knowledge related to platyhelminth VAL diversity across the phylum. Subsequent phylogenetic and tertiary structural analyses reveal several class-specific VAL features, which likely indicate a range of roles mediated by this protein family. Our comprehensive analysis of platyhelminth VALs represents a unifying synopsis for understanding diversity within this protein family and a firm context in which to initiate future functional characterization of these enigmatic members.

Background Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. Methodology/Principal Findings In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. Conclusion This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack., Author Summary Schistosomiasis is one of the major parasitic diseases in developing countries and still causes 200,000 deaths per year. Mass drug administration programs with praziquantel, the drug of choice against schistosomiasis, are currently undertaken in Sub-Saharan Africa. Praziquantel, although efficient against adult worms, fails to cure early infection. The complex developmental stages of schistosomes and migration through varying host compartments with different local drug concentration are a challenge for drug treatment. After infecting their mammalian host, schistosomula traverse through skin and the vasculature of lung, liver and intestines. During their migration, they develop from larvae to paired adults in approximately 4 to 5 weeks. So far, drug effects have been analyzed on adult worms or freshly transformed schistosomula only. Information about the effects on the larval stages is lacking. We were able to transfer the larval development of the first three weeks into the culture dish. This tool can be used for the analysis of drug effects against schistosomula and for investigation of the accessibility, expression and localization of antigens. Rendering the parasite’s larvae vulnerable to the host’s immune system by increasing antigen presentation is an important aspect of drug activity. We demonstrate on in vitro-cultured Schistosoma mansoni larvae, that SmCD59a and SmKK7, as examples for hidden antigens, become accessible to antibodies following drug treatment.

Fitzsimmons, C. M., Schramm, G., Jones, F. M., Chalmers, I. W., Hoffmann, K. F., Grevelding, C. G., Wuhrer, M., Hokke, C. H., Haas, H., Doenhoff, M. J., Dunne, D. W. (2005). Molecular characterization of omega-1: A hepatotoxic ribonuclease from Schistosoma mansoni eggs. Molecular & Biochemical Parasitology, 144(1), pp. 123-7.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

Graphical abstract, Highlights • Schistosoma mansoni VAL9 (SmVAL9) is a secreted N-linked glycoprotein containing a unique, difucosyl modification. • SmVAL9 is found throughout miracidia/sporocyst parenchymal cell inclusions/vesicles and germinal cells. • SmVAL9 differentially regulates murine and snail matrix metalloproteinases., The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.

Background The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and ‘Turbellaria’) contain methylated cytosines within their genome compartments. Conclusions Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.

Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain proteins mirrors the detection of cytosine methylation abundance and implicates the presence of a functional DNA methylation machinery. Genome losses in cytosine methylation upon SmDnmt2 silencing and the identification of a hypermethylated, repetitive intron within a predicted forkhead gene confirm this assertion. Importantly, disruption of egg production and egg maturation by 5-azacytidine establishes an essential role for 5-methylcytosine in this parasite. These findings provide the first functional confirmation for this epigenetic modification in any worm species and link the cytosine methylation machinery to platyhelminth oviposition processes., The chronic disease schistosomiasis is caused by the blood fluke Schistosoma mansoni. By studying DNA modifications throughout the lifecycle of the pathogen, the authors identify DNA methylation as a factor in egg development and suggest that the epigenetic machinery responsible may be a therapeutic target.

Background Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis. Methodology/Principal Findings Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons. Conclusions/Significance Collectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex., Author Summary Despite the implementation of focused and well-funded chemotherapeutic control initiatives over the last decade, schistosomiasis remains a significant cause of morbidity and mortality within countries of the developing world. There is, therefore, an urgent need for the rapid translation of genomic information into viable vaccines or new drug classes capable of eradicating the parasitic schistosome worms responsible for this neglected tropical disease. In our effort to identify potential targets for novel chemotherapeutic and immunoprophylactic interventions, we detail a combined bioinformatics approach, comprising exploratory statistical and network analyses, to thoroughly describe the transcriptional progression of Schistosoma mansoni across three environmental niches. Our results indicate that, although schistosomes are masters at host deception and survival, there are numerous exploitable candidate molecules displaying either differential or constitutive expression throughout the parasite's lifecycle. Importantly, some of these transcripts represent gene families not commonly found outside—or expanded within—the phylum Platyhelminthes, and thus represent priority targets. Many of the candidates identified herein will be subjected to ongoing and future hypothesis-led functional investigations. The completion of such specific examinations ultimately will contribute to the successful development of novel control strategies useful in the alleviation of schistosome-induced immunopathologies, morbidities and mortalities.

Schistosome helminths constitute a major health risk for the human population in many tropical areas. We demonstrate for the first time that several developmental stages of the human parasite Schistosoma mansoni express arginase, which is responsible for the hydrolysis of l-arginine to l-ornithine and urea. Arginase activity by alternatively activated macrophages is an essential component of the mammalian host response in schistosomiasis. However, it has not been previously shown that the parasite also expresses arginase when it is in contact with the mammalian host. After cloning and sequencing the cDNA encoding the parasite enzyme, we found that many structural features of human arginase are well conserved in the parasite ortholog. Subsequently, we discovered that S. mansoni arginase shares many similar molecular, biochemical and functional properties with both human arginase isoforms. Nevertheless, our data also reveal striking differences between human and schistosome arginase. Particularly, we found evidence that schistosome arginase activity depends upon disulphide bonds by cysteines, in contrast to human arginase. In conclusion, we report that S. mansoni arginase is well adapted to the physiological conditions that exist in the human host.

Background A human IgE response to Sm22.6 (a dominant IgE target in Schistosoma mansoni) is associated with the development of partial immunity. Located inside the tegument, the molecule belongs to a family of proteins from parasitic platyhelminths, the Tegument-Allergen-Like proteins (TALs). In addition to containing dynein-light-chain domains, these TALs also contain EF-hand domains similar to those found in numerous EF-hand allergens. Methodology/Principal Findings S. mansoni genome searches revealed 13 members (SmTAL1-13) within the species. Recent microarray data demonstrated they have a wide range of life-cycle transcriptional profiles. We expressed SmTAL1 (Sm22.6), SmTAL2, 3, 4, 5 and 13 as recombinant proteins and measured IgE and IgG4 in 200 infected males (7–60 years) from a schistosomiasis endemic region in Uganda. For SmTAL1 and 3 (transcribed in schistosomula through adult-worms and adult-worms, respectively) and SmTAL5 (transcribed in cercariae through adult-worms), detectable IgE responses were rare in 7–9 year olds, but increased with age. At all ages, IgE to SmTAL2 (expressed constitutively), was rare while anti-SmTAL2 IgG4 was common. Levels of IgE and IgG4 to SmTAL4 and 13 (transcribed predominantly in the cercariae/skin stage) were all low. Conclusions We have not measured SmTAL protein abundance or exposure in live parasites, but the antibody data suggests to us that, in endemic areas, there is priming and boosting of IgE to adult-worm SmTALs by occasional death of long-lived worms, desensitization to egg SmTALs through continuous exposure to dying eggs and low immunogenicity of larval SmTALs due to immunosuppression in the skin by the parasite. Of these, it is the gradual increase in IgE to the worm antigens that parallels age-dependent immunity seen in endemic areas., Author Summary Examining the genome of the parasitic worm Schistosoma mansoni, we have identified and defined the structure of a family of 13 allergen-like proteins from the outer layer (tegument) of the organism. We term these molecules the S. mansoni Tegument-Allergen-Like proteins (SmTALs). During S. mansoni infection the human host is exposed to skin-invading larvae, adult-worms (living in the blood) and to parasite eggs. These life-stages have very different sizes, tissue composition and gene expression. We have produced 6 SmTAL proteins with different life-cycle transcriptional patterns and measured IgE antibody responses to them in 200 infected males from an S. mansoni endemic area. The binding of IgE to foreign proteins is important in allergy but also in defence against parasitic worms. Our results suggest that, in these endemic areas, there is priming and boosting of IgE responses to adult-worm SmTALs by the occasional death of long-lived worms, desensitization to egg SmTALs due to continuous exposure to dying eggs and low immunogenicity of larval SmTALs perhaps due to immunosuppression in the skin by the parasite. Schistosome infection is a major health problem in many countries. Our work provides insight into what provokes and controls the antibody responses associated with human immunity to this parasite.

Background Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genome to drug’ lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. Methodology/Principal Findings We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative. Conclusions/Significance The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance., Author Summary With only one effective drug, praziquantel, currently used to treat most worldwide cases of schistosomiasis, there exists a pressing need to identify alternative anthelmintics before the development of praziquantel-resistant schistosomes removes our ability to combat this neglected tropical disease. At present, the most widely adopted methodology used to identify promising new anti-schistosome compounds relies on time consuming and subjective microscopic examination of parasite viability in response to in vitro schistosome/compound co-culturing. In our continued effort to identify novel drug and vaccine targets, we detail a dual-fluorescence bioassay that can objectively be used for assessing Schistosoma mansoni schistosomula viability in a medium or high- throughput manner to suit either academic or industrial settings. The described methodology replaces subjectivity with sensitivity and provides an enabling technology useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that results obtained from these quantifiable in vitro screens would prioritize the most effective anti-schistosomal compounds for follow-up in vivo experimentation. This highly-adaptable dual-fluorescence bioassay could be integrated with other methods for measuring schistosome phenotype and, together, be used to greatly accelerate our search for novel anthelmintics.

Background The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules. Proteins containing this domain are implicated in diverse biological activities and may be important for chronic host/parasite interactions. Results We report the first description of an SCP/TAPS gene family (Schistosoma mansoni venom allergen-like (SmVALs)) in the medically important Platyhelminthes (class Trematoda) and describe individual members' phylogenetic relationships, genomic organization and life cycle expression profiles. Twenty-eight SmVALs with complete SCP/TAPS domains were identified and comparison of their predicted protein features and gene structures indicated the presence of two distinct sub-families (group 1 & group 2). Phylogenetic analysis demonstrated that this group 1/group 2 split is zoologically widespread as it exists across the metazoan sub-kingdom. Chromosomal localisation and PCR analysis, coupled to inspection of the current S. mansoni genomic assembly, revealed that many of the SmVAL genes are spatially linked throughout the genome. Quantitative lifecycle expression profiling demonstrated distinct SmVAL expression patterns, including transcripts specifically associated with lifestages involved in definitive host invasion, transcripts restricted to lifestages involved in the invasion of the intermediate host and transcripts ubiquitously expressed. Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing. Conclusion Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa. The extensive lifecycle expression analysis indicates several SmVAL transcripts are upregulated in infective stages of the parasite, suggesting that these particular protein products may be linked to the establishment of chronic host/parasite interactions.