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Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Here we describe a new class of cryptides (peptides encrypted within a larger protein) with antimicrobial properties, named schistocins, derived from SmKI-1, a key protein in Shistosoma mansoni survival. This is a multi-functional protein with biotechnological potential usage as a therapeutic molecule in inflammatory diseases and to control schistosomiasis., Methods: We used our algorithm enCrypted, to perform an in silico proteolysis of SmKI-1 and a screening for potential antimicrobial activity. The selected peptides were chemically synthesized, tested in vitro and evaluated by both structural (CD, NMR) and biophysical (ITC) studies to access their structure-function relationship., Results: EnCrypted was capable of predicting AMPs in SmKI-1. Our biophysical analyses described a membrane-induced conformational change from random coil-to-α-helix and a peptide-membrane equilibrium for all schistocins. Our structural data allowed us to suggest a well-known mode of peptide-membrane interaction in which electrostatic attraction between the cationic peptides and anionic membranes results in the bilayer disordering. Moreover, the NMR H/D exchange data with the higher entropic contribution observed for the peptide-membrane interaction showed that schistocins have different orientations upon the membrane., Conclusions: This work demonstrate the robustness for using the physicochemical features of predicted peptides in the identification of new bioactive cryptides. Besides, it demonstrates the relevance of combining these analyses with biophysical methods to understand the peptide-membrane affinity and improve further algorithms., General Significance: Bioprospecting cryptides can be conducted through data mining of protein databases demonstrating the success of our strategy. The peptides-based agents derived from SmKI-1 might have high impact for system-biology and biotechnology., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Determining Schistosoma mansoni infection rate and intensity is challenging due to the low sensitivity of the Kato-Katz (KK) test that underestimates the true disease prevalence. Circulating cathodic antigen (CCA) excreted in urine is constantly produced by adult worms and has been used as the basis of a simple, non-invasive point of care test (POC-CCA) for Schistosoma mansoni infections. Although the abundance of CCA in urine is proportional to worm burden, the POC-CCA test is marketed as a qualitative test, making it difficult to investigate the wide range of infection intensities. This study was designed to compare the prevalence and intensity of S. mansoni by KK and POC-CCA and quantify, on fresh and frozen (<-20°C) urine samples, CCA using the visual scores and the ESEquant LR3 reader., Methodology: Stool and urine samples were collected from 759 school-aged children. The prevalence and intensity of S. mansoni were determined using KK and POC-CCA. The degree of the positivity of POC-CCA was estimated by quantifying CCA on fresh and frozen urine samples using visual scores and strip reader. The prevalence, the infection intensity as well the relative amounts of CCA were compared., Results: The S. mansoni infection rates inferred from POC-CCA and KK were 40.7% and 9.4% respectively. Good correlations were observed between infection intensities recorded by; i) the reader and visual scoring system on fresh (Rho = 0.89) and frozen samples (Rho = 0.97), ii) the reader on fresh urine samples and KK (epg) (Rho = 0.44). Nevertheless, 238 POC-CCA positive children were negative for KK, and sixteen of them had high levels of CCA. The correlation between results from the reader on fresh and frozen samples was good (Rho = 0.85). On frozen samples, CCA was not detected in 55 samples that were positive in fresh urine samples., Conclusion: This study confirmed the low sensitivity of KK and the high capacity of POC-CCA to provide reliable data on the prevalence and intensity of S. mansoni infections. The lateral flow reader enabled accurate quantification of CCA under field conditions on fresh and frozen urine samples with less time and effort than KK., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis is a disease of poverty affecting millions of people. Praziquantel (PZQ), with its strengths and weaknesses, is the only treatment available. We previously reported findings on three lead compounds derived from oxamniquine (OXA), an old antischistosomal drug: ferrocene-containing (Fc-CH 2 -OXA), ruthenocene-containing (Rc-CH 2 -OXA) and benzene-containing (Ph-CH 2 -OXA) OXA derivatives. These derivatives showed excellent in vitro activity against both Schistosoma mansoni larvae and adult worms and S. haematobium adult worms, and were also active in vivo against adult S. mansoni. Encouraged by these promising results, we conducted additional in-depth preclinical studies and report in this investigation on metabolic stability studies, in vivo studies on S. haematobium and juvenile S. mansoni, computational simulations, and formulation development. Molecular dynamics simulations supported the in vitro results on the target protein. Though all three compounds were poorly stable within an acidic environment, they were only slightly cleared in the in vitro liver model. This is likely the reason why the promising in vitro activity did not translate into in vivo activity on S. haematobium. This limitation could not be overcome by the formulation of lipid nanocapsules as a way to improve the in vivo activity. Further studies should focus on increasing the compound's bioavailability, to reach an active concentration in the microenvironment of the parasite., (© 2020 Wiley-VCH GmbH.)

The parasites and eggs of helminths, including schistosomes, are associated with factors that can modulate the nature and outcomes of host immune responses, particularly enhancing type 2 immunity and impairing the effects of type 1 and type 17 immunity. The main species of schistosomes that cause infection in humans are capable of generating a microenvironment that allows survival of the parasite by evasion of the immune response. Schistosome infections are associated with beneficial effects on chronic immune disorders, including allergies, autoimmune diseases, and alloimmune responses. Recently, there has been increasing research interest in the role of schistosomes in immunoregulation during human infection, and the mechanisms underlying these roles continue to be investigated. Further studies may identify potential opportunities to develop new treatments for immune disease. In this review, we provide an update on the advances in our understanding of schistosome-associated modulation of the cells of the innate and adaptive immune systems as well as the potential role of schistosome-associated factors as therapeutic modulators of immune disorders, including allergies, autoimmune diseases, and transplant immunopathology. We also discuss potential opportunities for targeting schistosome-induced immunoregulation for future translation to the clinical setting., (Copyright © 2020 American Society for Microbiology.)

Schistosomes are human pathogens causing the neglected tropical disease schistosomiasis, which occurs worldwide in (sub-)tropical regions. This infectious disease is often associated with poverty, and more than 700 million people are at risk of infection. Exploitation of novel habitats and limited therapeutic options brought schistosomes into research focus. Schistosomes are the only trematodes that have evolved separate sexes. They are covered by their metabolically active tegument, a surface area representing the interface between male and female in their permanent mating contact but also between parasite and host. The tegument comprises, besides others, numerous specific lipid compounds. Limited information is available on the exact lipid composition and its spatial distribution. We used atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to characterize the Schistosoma mansoni tegument surface in comparison to tissue sections of whole worms or couples. We found that phosphatidylcholines (PC) and specific phosphatidylethanolamines (PE) are significantly more abundant inside the worm body compared to the tegument. On the other hand, the latter was found to be enriched in sphingomyelins (SM), phosphatidylserines (PS), lysophosphatidylcholines (LPC), and specific PE species. We further investigated lipid classes concerning number of carbon atoms in fatty acyl chains as well as the degree of unsaturation and found pronounced differences between the tegument and whole-worm body. Furthermore, differences between male and female teguments were found. The lipid composition of S. mansoni tissues has been investigated in an untargeted, spatially resolved manner for the first time., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Bernhard Spengler and CGG are consultants of TransMIT GmbH Giessen.

Helminth parasites secrete extracellular vesicles (EVs) into their environment that have potential roles in host-parasite communication, and thus represent potentially useful targets for novel control strategies. Here, we carried out a comprehensive proteomic analysis of two different populations of EVs - 15k pellet and 120k pellet EVs - from Schistosoma mansoni adult worms. We characterised the proteins present in the membranes of the EVs (including external trypsin-liberated peptides, integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs)), as well as cargo proteins, using LC-MS/MS. A total of 286 and 716 proteins were identified in 15k and 120k pellets, respectively. Some of the most abundant proteins identified from both 15k and 120k pellets include known vaccine candidates such as Sm-TSP-2, saponin B domain-containing proteins, calpain glutathione-S-transferase, Sm29 and cathepsin domain-containing proteins. Other abundant proteins that have not been tested as vaccines include DM9 domain-containing protein, 13 kDa tegumental antigen and histone H4-like protein. Sm23, a member of the tetraspanin family with known vaccine efficacy, was identified in the cargo and IMP compartments of only 15k pellet vesicles. Moreover, a collection of proteins with known or potential relevance in host-parasite communication including proteases, antioxidants and EV biogenesis/trafficking of both vesicle types were identified. Our results provide the first report of a comprehensive compartmental proteomic analysis of adult S. mansoni-derived EVs. Future research should investigate recombinant forms of these proteins as vaccine and serodiagnostic antigens as well as the roles of EV proteins in host-parasite communication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model., Methods and Results: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected., Conclusion: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development., (© 2019 John Wiley & Sons Ltd.)

Background: Schistosomiasis (snail fever/bilharzia), a disease caused by parasitic flatworms (schistosomes), infects millions of people worldwide. Aquaporins from these organisms were found to be a potent drug target., Introduction: We investigate the possible mechanism of inhibition of Aquaporin (AQP) from S.mansoni by 5 drug molecules (Praziquantel, Metrifonate, Artimisinin, Albendazole, and Amoscanate)., Methods: 3D molecular structure of Aquaporin was obtained through homology modeling and further protein-ligand docking and MD simulation were performed., Results: VAL-75, ASN-91, ALA-220, ASN-222, ARG-225 amino acids were found to play crucial role in ligand binding. TRP-71 and other important residues play major role in hydrophobic interactions stabilizing protein-ligand complexes., Conclusion: We hope that this study (with the newly identified aquaporin target) will support the development of structure and pharmacophore-based novel S. mansoni drugs to control and curb Schistosomiasis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Currently, three recombinant antigens based vaccines are under clinical trials against Schistosomiasis, but there is no vaccine available for prophylaxis or therapeutic. This study was conducted to construct a multi-epitope based vaccine against Schistosoma mansoni via utilizing Sm14, Sm21.7, Sm23, Sm29, Smp80, Sm-CB and SM-TSP-2 antigens. Helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL) and IFN-γ epitopes were predicted. Furthermore, Pan HLA DR-binding epitope was added to the vaccine. Moreover, 50S ribosomal protein L7/L12 of Mycobacterium tuberculosis as a novel TLR4 agonist was applied. The TAT peptide was added to the vaccine to augment intracellular delivery. The selected epitopes were linked together through appropriate linkers and chimeric vaccine was constructed with 617 amino acids with molecular weight of 65.43 kDa. Physico-chemical properties revealed a soluble protein with antigenic and non-allergic properties. Further analyses validated the stability of the construct that was able to interact with TLR4. Immunoinformatics analysis demonstrated the strong potential of constructed vaccine to stimulate T and B-cell mediated immune responses. In summary, obtained data indicated that the proposed vaccine can properly induce both T and B cells immune responses and could possibly be utilized for prophylactic or therapeutic aims in response to infection caused by S. mansoni., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Schistosomes are parasitic platyhelminthes that cause schistosomiasis, which is a life-threatening infectious disease for humans in the tropics and subtropics worldwide. Within the human host, female and male schistosomes develop and pair as a prerequisite for egg production. Part of the eggs get lodged in organs such as the gut, spleen, and liver, where they cause severe inflammatory processes, including liver fibrosis, which is one of the most serious pathological symptoms. High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) has been used as a powerful tool to investigate adult schistosomes at the topographic molecular level. An MSI-compatible protocol was developed, covering critical sample preparation steps and focusing on obtaining artifact-free, longitudinal cryosections. Planar, consecutive sections were prepared from ∼400 μm thick S. mansoni worm couples, comparing several microembedding approaches. High-resolution MSI at both, 10 and 5 μm lateral resolution unraveled anatomical structures and differential abundances of glycerophospholipids and saccharides in females and males. In addition, glycerophospholipids occurred differentially abundant in worm tissues of the female, such as the gut, which is essential for nutrient uptake and subsequent metabolism. Fragment ions of isobaric phospholipids were investigated by on-tissue MS 2 imaging experiments, unambiguously showing isomer-specific ion signals. This study provides a solid basis for investigating schistosome parasites in chemical detail at the whole-worm level by MSI.

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Six dibenzylbutyrolactonic lignans ((-)-hinokinin (1), (-)-cubebin (2), (-)-yatein (3), (-)-5-methoxyyatein (4), dihydrocubebin (5) and dihydroclusin (6)) were isolated from Piper cubeba seed extract and evaluated against Schistosoma mansoni. All lignans, except 5, were able to separate the adult worm pairs and reduce the egg numbers during 24 h of incubation. Lignans 1, 3 and 4 (containing a lactone ring) were the most efficient concerning antiparasitary activity. Comparing structures 3 and 4, the presence of the methoxy group at position 5 appears to be important for this activity. Considering 1 and 3, it is possible to see that the substitution pattern change (methylenedioxy or methoxy groups) in positions 3' and 4' alter the biological response, with 1 being the second most active compound. Computational calculations suggest that the activity of compound 4 can be correlated with the largest lipophilicity value., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

Accurate and sensitive point-of-care diagnostic tools are critical for schistosomiasis control and elimination. The existing ultrasensitive lateral flow assay for the detection of Schistosoma circulating anodic antigen (CAA) has demonstrated excellent sensitivity but is time-consuming and requires significant laboratory infrastructure that limits its applicability at the point of care. To address this challenge, we sought to develop an alternative sample preparation method to concentrate CAA from large-volume urine samples requiring little-to-no laboratory equipment. The developed method relies on electrostatic interactions between the negatively-charged CAA biomarker and positively-charged poly(amidoamine) (PAMAM) dendrimers functionalized to the surface of magnetic particles. After CAA capture on the surface of the PAMAM-functionalized magnetic beads, the supernatant was removed, and CAA was eluted into a small-volume, high-salt elution buffer. This concentrated eluate was subsequently applied to the existing lateral flow assay. The PAMAM-functionalized magnetic bead-based CAA concentration method was extensively characterized for its robustness, evaluated on a set of endemic urine samples, and compared to spin filter-based concentration methods. The novel bead-based sample preparation method used only disposable laboratory materials, resulted in a 200-fold improvement in CAA limits of detection, and performed just as well as infrastructure-intensive and high-cost spin filter methods. Additionally, the functionalized beads were robust to variations in sample pH and storage conditions. The PAMAM-functionalized magnetic bead-based CAA concentration method represents a promising step toward ultrasensitive schistosomiasis diagnosis at the point of care.

Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity., Competing Interests: The authors have declared that no competing interests exist.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Schistosoma mansoni, like other trematodes, expresses a number of unusual calcium binding proteins which consist of an EF-hand domain joined to a dynein light chain-like (DLC-like) domain by a flexible linker. These proteins have been implicated in host immune responses and drug binding. Three members of this protein family from S. mansoni (SmTAL1, SmTAL2 and SmTAL3) have been well characterised biochemically. Here we characterise the remaining family members from this species (SmTAL4-13). All of these proteins form homodimers and all except SmTAL5 bind to calcium and manganese ions. SmTAL9, 10 and 11 also bind to magnesium ions. The antischistosomal drug, praziquantel interacts with SmTAL4, 5 and 8. Some family members also bind to calmodulin antagonists such as chlorpromazine and trifluoperazine. Molecular modelling suggests that all ten proteins adopt similar overall folds with the EF-hand and DLC-like domains folding discretely. Bioinformatics analyses suggest that the proteins may fall into two main categories: (i) those which bind calcium ions reversibly at the second EF-hand and may play a role in signalling (SmTAL1, 2, 8 and 12) and (ii) those which bind calcium ions at the first EF-hand and may play either signalling or structural roles (SmTAL7, 9, 10 and 13). The remaining proteins include those which do not bind calcium ions (SmTAL3 and 5) and three other proteins (SmTAL4, 6 and 11). The roles of these proteins are less clear, but they may also have structural roles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glutamate-gated chloride channels (GluCls) are neurotransmitter receptors that mediate crucial inhibitory signaling in invertebrate neuromuscular systems. Their role in invertebrate physiology and their absence from vertebrates make GluCls a prime target for antiparasitic drugs. GluCls from flatworm parasites are substantially different from and are much less understood than those from roundworm and insect parasites, hindering the development of potential therapeutics targeting GluCls in flatworm-related diseases such as schistosomiasis. Here, we sought to dissect the molecular and chemical basis for ligand recognition in the extracellular glutamate binding site of SmGluCl-2 from Schistosoma mansoni, using site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings. Our results indicate that aromatic residues in ligand binding loops A, B, and C are important for SmGluCl-2 function. Loop C, which differs in length compared to other pentameric ligand-gated ion channels (pLGICs), contributes to ligand recognition through both an aromatic residue and two vicinal threonine residues. We also show that, in contrast to other pLGICs, the hydrophobic channel gate in SmGluCl-2 extends from the 9' position to the 6' position in the channel-forming M2 helix. The 6' and 9' positions also seem to control sensitivity to the pore blocker picrotoxin.

Some RNA classes require folding into the proper higher-order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA-binding ligand, naphthyridine carbamate tetramer with Z-stilbene linker (Z-NCTS). Binding of Z-NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z-NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z-NCTS-responsive genetic switch using the HHR sequence inserted into the 3' untranslated region as a cis-acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Schistosoma mansoni venom allergen-like protein (SmVAL) is a gene family composed of 29 members divided into group 1 encoding proteins potentially secreted, and group 2 encoding intracellular components. Some members were found to be upregulated in the transition of germ ball - cercariae - day 3 schistosomula, suggesting that group 1 SmVAL proteins are associated with the invasion of the human host, although their functions are not completely established. Recently, we have described the localization of SmVAL7 (group 1) and SmVAL6 (group 2) transcripts in the oesophageal gland and in the oral and ventral suckers of adult parasites, respectively. The expression patterns of the two genes suggest that SmVAL7 protein plays a role in the blood-feeding process while SmVAL6 is associated with the parasite attachment and movement in the vasculature. In this way, searching for additional secreted SmVAL proteins that could be involved in key processes from skin penetration to the beginning of blood-feeding, we investigated the tissue localization of SmVAL4, 13, 16 and 24 by whole-mount in situ hybridization (WISH)., Results: We report here the localization of group 1 SmVAL4 and 24 transcripts in the pre-acetabular glands of developing germ balls. Time course experiments of in vitro cultured schistosomula after cercariae transformation demonstrated that SmVAL4 protein is secreted during the first 3 h of in vitro culture, correlating with the emptying of acetabular glands as documented by confocal microscopy. In addition, the localization of SmVAL13 transcripts in adult male anterior oesophageal gland suggests that the respective protein may be involved in the first steps of the blood-feeding process. SmVAL16 was localized close to the neural ganglia and requires further investigation., Conclusions: Our findings demonstrate that SmVAL proteins have localizations that place them in strategic positions to be considered as potential vaccine candidates as some members are exposed to interaction with the immune system and may participate in key processes of mammalian invasion and parasitism establishment.

In the context of investigating the exceptional reproductive biology of schistosomes, several studies have focused on the identification and characterization of involved molecules. Among these are cellular tyrosine kinases (CTKs) which control differentiation processes in the female gonads. On the way to unravel CTK-mediated signaling processes in more detail, several upstream- and downstream partners of these CTKs were identified. In this context we present here first data characterizing the novel orphan gene Sm opg1. Annotated as hypothetical protein, SmOPG1 was identified as an interaction partner of the CTK SmTK6 by yeast two-hybrid library screening. Y2/3H interaction studies showed that SmTK6 binds with its SH2 domain to a specific binding motif within the C-terminus of SmOPG1. Additionally, in situ-hybridization and organ-specific RT-PCR analyses demonstrated the co-localization of SmOPG1 and SmTK6 transcripts in the gonads of adult S. mansoni. Finally, SmOPG1 knock-down provided first hints for a function in the ovary., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

Serine protease inhibitors, known as serpins, are pleiotropic regulators of endogenous and exogenous proteases, and molecule transporters. They have been documented in animals, plants, fungi, bacteria, and viruses; here, we characterize a serpin from the trematode platyhelminth Schistosoma mansoni. At least eight serpins have been found in the genome of S. mansoni, but only two have characterized molecular properties and functions. Here, the function of S. mansoni serpin isoform 3 (SmSPI) was analyzed, using both computational and molecular biological approaches. Phylogenetic analysis showed that SmSPI was closely related to Schistosoma haematobium serpin and Schistosoma japonicum serpin B10. Structure determined in silico confirmed that SmSPI belonged to the serpin superfamily, containing nine α-helices, three β-sheets, and a reactive central loop. SmSPI was highly expressed in schistosomules, predominantly in the head gland, and in adult male and female with intensive accumulation on the spines, which suggests that it may have a role in facilitating intradermal and intravenous survival. Recombinant SmSPI was overexpressed in Escherichia coli; the recombinant protein was of the same size (46 kDa) as the native protein. Immunological analysis suggested that mice infected with S. mansoni responded to rSmSPI at 8 weeks postinfection (wpi) but not earlier. The inhibitory activity of rSmSPI was specific to chymotrypsin but not trypsin, neutrophil elastase, and porcine pancreatic elastase. Elucidating the biological and physiological functions of SmSPI as well as other serpins will lead to further understanding of host-parasite interaction machinery that may provide novel strategies to prevent and control schistosomiasis in the future.

An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A proteome microarray consisting of 992 Schistosoma mansoni proteins was produced and screened with sera to determine antibody signatures indicative of the clinical stages of schistosomiasis and the identification of subunit vaccine candidates. Herein, we describe the methods used to derive the gene list for this array (representing approximately 10% of the predicted S. mansoni proteome). We also probed a pilot version of the microarray with sera from individuals either acutely or chronically infected with S. mansoni from endemic areas in Brazil and sera from individuals resident outside the endemic area (USA) to determine if the array is functional and informative., (Copyright © 2016. Published by Elsevier Ltd.)

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins., (Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Schistosomiasis is the most important human helminth infection due to its impact on public health. The clinical manifestations are chronic and significantly decrease an individual's quality of life. Infected individuals suffer from long-term organ pathologies including fibrosis which eventually leads to organ failure. The development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission., Method: Our group has chosen Schistosoma mansoni (Sm) cathepsin B, a peptidase involved in parasite feeding, as a prospective vaccine candidate. Our experimental formulation consisted of recombinant Sm-cathepsin B formulated in Montanide ISA 720 VG, a squalene based adjuvant containing a mannide mono-oleate emulsifier. Parasitological burden was assessed by determining adult worm, hepatic egg, and intestinal egg numbers in each mouse. Serum was used in ELISAs to evaluate production of antigen-specific antibodies, and isolated splenocytes were stimulated with the antigen for the analysis of cytokine secretion levels., Results: The Sm-cathepsin B and Montanide formulation conferred protection against a challenge infection by significantly reducing all forms of parasitological burdens. Worm burden, hepatic egg burden and intestinal egg burden were decreased by 60%, 6%, and 56%, respectively in immunized animals compared to controls (P = 0.0002, P < 0.0001, P = 0.0009, respectively). Immunizations with the vaccine elicited robust production of Sm-cathepsin B specific antibodies (endpoint titers = 122,880). Both antigen-specific IgG1 and IgG2c titers were observed, with the former having more elevated titers. Furthermore, splenocytes isolated from the immunized animals, compared to control animals, secreted higher levels of key Th1 cytokines, IFN-γ, IL-12, and TNF-α, as well as the Th2 cytokines IL-5 and IL-4 when stimulated with recombinant Sm-cathepsin B. The Th17 cytokine IL-17, the chemokine CCL5, and the growth factor GM-CSF were also significantly increased in the immunized animals compared to the controls., Conclusion: The formulation tested in this study was able to significantly reduce all forms of parasite burden, stimulate robust production of antigen-specific antibodies, and induce a mixed Th1/Th2 response. These results highlight the potential of Sm-cathepsin B/Montanide ISA 720 VG as a vaccine candidate against schistosomiasis.

Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases.

Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Schistosoma mansoni causes intestinal schistosomiasis, a disease that is prevalent in several regions worldwide. To date, a protective vaccine against S. mansoni is still lacking. Several promising antigens have been discovered and evaluated for vaccine protection, such as Sm14 and Sm28GST. In this short communication, we report the successful detection of an alternatively spliced truncated form of Sm14 which was highly expressed in an Egyptian strain of S. mansoni. This truncated Sm14 (TrSm14) protein was formerly reported to be practically non-existent and its complementary DNA (cDNA) was thought to be 'a rare misprocessing of mRNA precursor'. Our finding demonstrates that there is inter-strain variation in the S. mansoni transcriptome and subsequently in the role/function of the expressed proteins. We expressed TrSm14 successfully in Escherichia coli as a fusion protein with the schistosomal antigen Sm28GST. The fusion protein was purified using metal affinity chromatography and was found to be reactive with serum from S. mansoni-infected patients. This suggests a possible diagnostic value for this protein in detection of anti-schistosomal antibodies. In addition, this fusion protein could offer a potential bivalent vaccine candidate against S. mansoni that is worthy of further investigation.

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Praziquantel (PZQ) is the treatment of choice for schistosomiasis, one of the most important but neglected tropical diseases. Recently, however, Schistosoma have exhibited reduced susceptibility to PZQ, and an urgent need to develop new drugs to treat schistosomiasis has emerged. Thioredoxin glutathione reductase (TGR) plays a crucial role in the redox balance of the parasite, combining glutaredoxin (Grx), glutathione reductase and thioredoxin reductase (TR) activities. Several compounds, including oxadiazole 2‑oxides, phosphinic acid amides, isoxazolones and phosphoramidites, have been identified as agents that inhibit TGR from Schistosoma mansoni (smTGR) and exhibit anti‑schistosomal activity. 4‑Phenyl‑1,2,5‑oxadiazole‑3‑carbonitrile‑2‑oxide has also been shown to be active against TGR from Schistosoma japonicum (sjTGR). The binding sites of these inhibitors, however, remain unclear. To explore the binding interactions of these compounds, we selected six compounds to dock into the NADPH binding site, the active site of the TR domain and the Grx active site of both smTGR and sjTGR using AutoDock 4.2.5.1. The results suggested that the most favoured binding site for all compounds in either sjTGR or smTGR was the oxidised glutathione‑binding pocket of the TR domain. Although all of the compounds could fit into the sjTGR site, the inhibition efficiency of these compounds towards sjTGR was marginally lower than it was towards smTGR, suggesting that it would be necessary to design specific inhibitors of TGR for different Schistosoma species. The docking results showed that all compounds docking in smTGR and sjTGR adopted similar binding modes in the TR domain. Two peptide fragments from another subunit, Phe505'‑Leu508' and Pro572'‑Thr577', played a critical role in the interactions with the inhibitors. In conclusion, the present study has revealed binding mechanisms for potential inhibitors of Schistosoma TGRs and could lead to structure‑based ligand design and the development of new anti-schistosomiasis drugs.

Background: The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored., Methodology/principal Findings: Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively) when compared to rising IgG1 levels against sub-surface SmTAL1., Conclusions/significance: Collectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

The tegument of Schistosoma mansoni plays an integral role in host-parasite interactions, particularly during the transition from the free-living cercariae to the intra-mammalian schistosomula stages. This developmental period is characterised by the transition from a trilaminate surface to a heptalaminate tegument that plays key roles in immune evasion, nutrition and excretion. Proteins exposed at the surface membranes of newly transformed schistosomula are therefore thought to be prime targets for the development of new vaccines and drugs for schistosomiasis. Using a combination of tegumental labelling and high-throughput quantitative proteomics, more than 450 proteins were identified on the apical membrane of S. mansoni schistosomula, of which 200 had significantly regulated expression profiles at different stages of schistosomula development in vitro, including glucose transporters, sterols, heat shock proteins, antioxidant enzymes and peptidases. Current vaccine antigens were identified on the apical membrane (Sm-TSP-1, calpain) or sub-tegumental (Sm-TSP-2, Sm29) fractions of the schistosomula, displaying localisation patterns that, in some cases, differ from that in the adult stage fluke. This work provides the first known in-depth proteomic analysis of the surface-exposed proteins in the schistosomula tegument, and some of the proteins identified are clear targets for the generation of new vaccines and drugs against schistosomiasis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans.

Background: The ubiquitination process can be reversed by deubiquitinating enzymes (DUBs). These proteases are involved in ubiquitin processing, in the recovery of modified ubiquitin trapped in inactive forms, and in the recycling of ubiquitin monomers from polyubiquitinated chains. The diversity of DUB functions is illustrated by their number and variety of their catalytic domains with specific 3D architectures. DUBs can be divided into five subclasses: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs or UBPs), ovarian tumour proteases (OTUs), Machado-Joseph disease proteases (MJDs) and JAB1/MPN/Mov34 metalloenzymes (JAMMs)., Methods: Considering the role that the ubiquitin-proteasome system has been shown to play during the development of Schistosoma mansoni, our main goal was to identify and characterize SmUSPs. Here, we showed the identification of putative ubiquitin-specific proteases using bioinformatic approaches. We also evaluated the gene expression profile of representative USP family members using qRT-PCR., Results: We reported 17 USP family members in S. mansoni that present a conservation of UCH domains. Furthermore, the putative SmUSP transcripts analysed were detected in all investigated stages, showing distinct expression during S. mansoni development. The SmUSPs exhibiting high expression profiles were SmUSP7, SmUSP8, SmUSP9x and SmUSP24., Conclusion: S. mansoni USPs showed changes in expression levels for different life cycle stages indicating their involvement in cellular processes required for S. mansoni development. These data will serve as a basis for future functional studies of USPs in this parasite.

Background: The soluble antigen preparation of adult schistosomes (SWAP) has often been used to probe host responses against these blood-dwelling parasites. Despite its long-established use there is limited knowledge about its composition. The information we provide here on the molecular composition of SWAP may contribute as a guide for a rational selection of antigenic targets., Methods: Label-free quantitative shotgun proteomics was employed to determine the composition and abundance of SWAP constituents. Briefly, paired adult Schistosoma mansoni worms were sonicated in PBS pH 7.2 and ultracentrifuged for recovery of the soluble supernatant. An aliquot was subjected to trypsin digestion. Resulting peptides were separated under ultra-high performance liquid chromatography and analysed using an orbitrap mass spectrometer. Spectral data were interrogated using SequestHT against an in-house S. mansoni database. Proteins were quantified by recording the mean area under curve obtained for up to three most intense detected peptides. Proteins within the 90(th) percentile of the total SWAP mass were categorized according to their sub-cellular/tissue location., Results: In this work we expanded significantly the list of known SWAP constituents. Through application of stringent criteria, a total of 633 proteins were quantitatively identified. Only 18 proteins account to 50% of the total SWAP mass as revealed by their cumulative abundance. Among them, none is predicted as a secreted molecule reinforcing the point that SWAP is dominated by cytosolic and cytoskeletal proteins. In contrast, only 3% of the mass comprised proteins proposed to function at the host-parasite interfaces (tegument and gut), which could conceivably represent vulnerable targets of a protective immune response. Paradoxically, at least 11 SWAP proteins, comprising ~25% of its mass, have been tested as vaccine candidates., Conclusions: Our data suggest that use of SWAP to probe host responses has greatest value for diagnostic purposes or assessing intensity of infection. However, the preparation is of limited utility as an antigen source for investigating host responses to proteins expressed at or secreted from worm-host interfaces. The data also pose the question as to why vaccination with SWAP, containing so many proposed vaccine candidates, has no additive or even synergistic effects on the induction of protection.

Schistosomes, like many eukaryotic pathogens, typically display morphologically distinct stages during their life cycles. Epigenetic mechanisms underlie the pathogens' morphological transformations, and the targeting of epigenetics-driven cellular programs therefore represents an Achilles' heel of parasites. To speed up the search for new antiparasitic agents, drugs validated for other diseases can be rationally optimized into antiparasitic therapeutics. Specifically, zinc-dependent histone deacetylases (HDACs) are the most explored targets for epigenetic therapies, notably for anticancer treatments. This review focuses on the development of drug-leads inhibiting HDACs from schistosomes. More precisely, current progress on Schistosoma mansoni HDAC8 (smHDAC8) provided a proof of concept that targeting epigenetic enzymes is a valid approach to treat diseases caused by schistosomes, and possibly other eukaryotic pathogens.

Schistosomiasis, caused by S. mansoni, is a tropical disease that affects over 200 million people worldwide. A novel approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during their life cycle. We recently identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here we present results from a virtual screening campaign on smHDAC8. Besides hydroxamates, several sulfonamide-thiazole derivatives were identified by a target-based virtual screening using a homology model of smHDAC8. In vitro testing of 75 compounds identified 8 hydroxamates as potent and lead-like inhibitors of the parasitic HDAC8. Solving of the crystal structure of smHDAC8 with two of the virtual screening hits confirmed the predicted binding mode.

Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4(+) T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/β-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn(2+) in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

Schistosomiasis is a chronically debilitating disease caused by parasitic worms of the genus Schistosoma, and it is a global problem affecting over 240 million people. Little is known about the regulatory proteins and mechanisms that control schistosome host invasion, gene expression, and development. Schistosome larvae, cercariae, are transiently free-swimming organisms and infectious to man. Cercariae penetrate human host skin directly using proteases that degrade skin connective tissue. These proteases are secreted from anucleate acetabular glands that contain many proteins, including heat shock proteins. Heat shock transcription factors are strongly conserved activators that play crucial roles in the maintenance of cell homeostasis by transcriptionally regulating heat shock protein expression. In this study, we clone and characterize the schistosome Heat shock factor 1 gene (SmHSF1). We verify its ability to activate transcription using a modified yeast one-hybrid system, and we show that it can bind to the heat shock binding element (HSE) consensus DNA sequence. Our quantitative RT-PCR analysis shows that SmHSF1 is expressed throughout several life-cycle stages from sporocyst to adult worm. Interestingly, using immunohistochemistry, a polyclonal antibody raised against an Hsf1-peptide demonstrates a novel localization for this conserved, stress-modulating activator. Our analysis suggests that schistosome Heat shock factor 1 may be localized to the acetabular glands of infective cercariae.