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Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both oxidized thioredoxin and glutathione with electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH). SmTGR is a drug target for the treatment of Schistosomiasis, an infection caused by Schistosoma platyhelminths residing in the blood vessels of the host. Schistosoma spp. are reliant on TGR enzymes as they lack catalase and so use reduced thioredoxin and glutathione to regenerate peroxiredoxins consumed in the detoxification of reactive oxygen species. SmTGR is a flavin adenine dinucleotide (FAD)-dependent enzyme, and we have used the flavin as a spectrophotometric reporter to observe the movement of electrons within the enzyme. The data show that NADPH fractionally reduces the active site flavin with an observed rate constant estimated in this study to be ∼3000 s -1 . The flavin then reoxidizes by passing electrons at a similar rate to the proximal Cys159-Cys154 disulfide pair. The dissociation of NADP + occurs with a rate of ∼180 s -1 , which induces the deprotonation of Cys159, and this coincides with the accumulation of an intense FAD-thiolate charge transfer band. It is proposed that the electrons then pass to the Cys596-Cys597 disulfide pair of the associated subunit in the dimer with a net rate constant of ∼2 s -1 . (Note: Cys597 is Sec597 in wild-type (WT) SmTGR.) From this position, the electrons can be passed to oxidized thioredoxin or further into the protein to reduce the Cys28-Cys31 disulfide pair of the originating subunit of the dimer. From the Cys28-Cys31 center, electrons can then pass to oxidized glutathione that has a binding site directly adjacent.

Fragment screening is a powerful drug discovery approach particularly useful for enzymes difficult to inhibit selectively, such as the thiol/selenol-dependent thioredoxin reductases (TrxRs), which are essential and druggable in several infectious diseases. Several known inhibitors are reactive electrophiles targeting the selenocysteine-containing C-terminus and thus often suffering from off-target reactivity in vivo . The lack of structural information on the interaction modalities of the C-terminus-targeting inhibitors, due to the high mobility of this domain and the lack of alternative druggable sites, prevents the development of selective inhibitors for TrxRs. In this work, fragments selected from actives identified in a large screen carried out against Thioredoxin Glutathione Reductase from Schistosoma mansoni (SmTGR) were probed by X-ray crystallography. SmTGR is one of the most promising drug targets for schistosomiasis, a devastating, neglected disease. Utilizing a multicrystal method to analyze electron density maps, structural analysis, and functional studies, three binding sites were characterized in SmTGR: two sites are close to or partially superposable with the NADPH binding site, while the third one is found between two symmetry related SmTGR subunits of the crystal lattice. Surprisingly, one compound bound to this latter site stabilizes, through allosteric effects mediated by the so-called guiding bar residues, the crucial redox active C-terminus of SmTGR, making it finally visible at high resolution. These results further promote fragments as small molecule probes for investigating functional aspects of the target protein, exemplified by the allosteric effect on the C-terminus, and providing fundamental chemical information exploitable in drug discovery.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1 - 5 are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1 - 5 , 7 , and 8 displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds 1 - 4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds 4 - 6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

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.

Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.

Background: Schistosomiasis, a parasitic disease also known as bilharzia and snail fever, is caused by different species of flatworms, such as Schistosoma mansoni (S. mansoni). Thioredoxin glutathione reductase (TGR) from S. mansoni (SmTGR) is a well-characterized drug target for schistosomiasis, yet no anti-SmTGR compounds have reached clinical trials, suggesting that therapeutic development against schistosomiasis might benefit from additional scaffolds targeting this enzyme., Methods: A high-throughput screening (HTS) assay in vitro against SmTGR was developed and applied to a diverse compound library. SmTGR activity was quantified with ThioGlo®, a reagent that fluoresces upon binding to the free sulfhydryl groups of the reaction product GSH (reduced glutathione)., Results: We implemented an HTS effort against 59,360 synthetic compounds. In the primary screening, initial hits (928 or 1.56 %) showing greater than 90 % inhibition on SmTGR activity at a final concentration of 10 μM for each compound were identified. Further tests were carried out to confirm the effects of these hits and to explore the concentration-dependent response characteristics. As a result, 74 of them (0.12 %) representing 17 chemical scaffolds were confirmed and showed a great concentration-dependent inhibitory trend against SmTGR, including structures previously shown to be lethal to schistosomal growth. Of these, two scaffolds displayed a limited structure-activity relationship. When tested in cultured larvae, 39 compounds had cidal activity in 48 h, and five of them killed larvae completely at 3.125 μM. Of these, three compounds also killed adult worms ex vivo at concentrations between 5 μM and 10 μM., Conclusion: These confirmed hits may serve as starting points for the development of novel therapeutics to combat schistosomiasis.

Investigations regarding the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives revealed 3-phenoxymethyl menadiones as a novel anti-schistosomal chemical series. These newly synthesized compounds (1-7) and their difluoromethylmenadione counterparts (8, 9) were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol methyl ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione derivative (7) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compounds tested was active in vivo, suggesting that the limited bioavailability may compromise compound activity. Therefore, future studies will be directed toward improving pharmacokinetic properties and bioavailability., (© 2015 FEBS.)

A series of NO-donor praziquantel hybrid compounds was obtained by combining praziquantel (PZQ) and furoxan moieties in a single entity. NO-donor properties of the furoxan derivatives were evaluated by detecting nitrite after incubation of the products in 7.4 pH buffered solution in the presence of L-cysteine. Structurally-related furazans, devoid of NO release capacity, were also synthesized for control purposes. All products were studied for their ability to inhibit recombinant Schistosoma mansoni thioredoxin glutathione reductase (TGR). Mobility and death of adult Schistosoma mansoni worms cultured in the presence of the products were evaluated versus PZQ. Analysis of the results showed that some products were endowed with both PZQ and NO-dependent antiparasitic properties. Compounds 6, 7, 18, and 24 emerged as the most interesting balanced hybrids, worthy of additional study on PZQ-resistant parasites., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest in the context of the emerging resistance to praziquantel in schistosomiasis therapy.

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

Improving the solubility of polysubstituted 1,4-naphthoquinone derivatives was achieved by introducing nitrogen in two different positions of the naphthoquinone core, at C-5 and at C-8 of menadione through a two-step, straightforward synthesis based on the regioselective hetero-Diels-Alder reaction. The antimalarial and the antischistosomal activities of these polysubstituted aza-1,4-naphthoquinone derivatives were evaluated and led to the selection of distinct compounds for antimalarial versus antischistosomal action. The Ag(II)-assisted oxidative radical decarboxylation of the phenyl acetic acids using AgNO(3) and ammonium peroxodisulfate was modified to generate the 3-picolinyl-menadione with improved pharmacokinetic parameters, high antimalarial effects and capacity to inhibit the formation of β-hematin.

2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATP-independent chaperones upon exposure to high peroxide concentrations. These alternative functions have been associated with changes in the oligomerization state from low-(LMW) to high-molecular-weight (HMW) species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer formed by two stacked decamers. The latter is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H(2)O(2) concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Schistosomiasis affects more than 200 million people globally. The pathology of schistosome infections is due to chronic tissue inflammation and damage from immune generated granulomas surrounding parasite eggs trapped in host tissues. Schistosoma species are unique among trematode parasites because they are dioecious; females require paring with male parasites in order to attain reproductive maturity and produce viable eggs. Ex vivo cultured females lose the ability to produce viable eggs due to an involution of the vitellarium and loss of mature oocytes. In order to better understand schistosome reproductive biology we used data generated by serial analysis of gene expression (SAGE) to identify uncharacterized genes which have different transcript abundance in mature females, those that have been paired with males, and immature females obtained from unisexual infections. To characterize these genes we used bioinformatics, transcript localization, and transcriptional analysis during the regression of in vitro cultured females. Genes transcribed exclusively in mature females localize primarily in the vitellocytes and/or the ovary. Genes transcribed exclusively in females from single sex infections localize to vitellocytes and subtegumental cells. As female reproductive tissues regress, eggshell precursor proteins and genes involved in eggshell synthesis largely have decreased transcript abundance. However, some genes with elevated transcript abundance in mature adults have increased gene expression following regression indicating that the genes in this study function both in eggshell biology as well as vitellogenesis and maintenance of female reproductive tissues. In addition, we found that genes enriched in females from single sex infections have increased expression during regression in ex vivo females. By using these transcriptional analyses we can direct research to examine the areas of female biology that are both relevant to understanding the overall process of female development and worm pairing while determining novel therapeutic approaches directed at the maturation of female schistosomes.

Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes-termed Smvlg1, Smvlg2, and Smvlg3-were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases.

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.

The genome sequence for Schistosoma mansoni has been determined, allowing the complete protein complement to be predicted. However, few functional genomics techniques have been developed for use in S. mansoni, limiting the usefulness of the sequence data. Here we describe a whole mount in situ hybridization (WISH) method that can be used to identify the tissue-specific expression of transcripts in S. mansoni. Using this protocol we determine the tissue-specific expression of tetraspanin 2, a female-enriched tetraspanin, phenol oxidase, the secretory Cu/Zn superoxide dismutase, and an Argonaute family member. The localization of these transcripts by WISH correlates with prior studies performed using immunohistochemistry and/or in situ hybridization on tissue sections. WISH can be adapted to screen multiple transcripts, thus identifying novel targets for drugs or vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS). Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS). The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism.

Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, and the process is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins were expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic-and zinc-coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities toward these substrates. Using real-time polymerase-chain reaction (RT-PCR), both proteins were found to be expressed in all stages of the parasite's life cycle, with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.

To identify gene(s) that may be associated with resistance/susceptibility in the intermediate snail host Biomphalaria glabrata to Schistosoma mansoni infection, a snail albumen gland cDNA library was differentially screened and a partial cDNA encoding an antioxidant enzyme thioredoxin peroxidase (Tpx), or peroxiredoxin (Prx), was identified. The 753bp full-length, single-copy, constitutively expressed gene now referred to as BgPrx4 was later isolated. BgPrx4 is a 2-Cys peroxiredoxin containing the conserved peroxidatic cysteine (C(P)) in the N-terminus and the resolving cysteine (C(R)) in the C-terminus. Sequence analysis of BgPrx4 from both resistant and susceptible snails revealed the presence of several (at least 7) single nucleotide polymorphisms (SNPs). Phylogenetic analysis indicated BgPrx4 to resemble a homolog of human peroxiredoxin, PRDX4. Northern analysis of hepatopancreas RNA from both resistant and susceptible snails showed that upon parasite exposure there were qualitative changes in gene expression. Quantitative real-time RT-PCR analysis showed differences in the levels of BgPrx4 transcript induction following infection, with the transcript up-regulated in resistant snails during the early phase (5h) of infection compared to susceptible snails in which it was down-regulated within the early time period. While there was an increase in transcription in susceptible snails later (48h) post-infection, this never reached the levels detected in resistant snails. A similar trend - higher, earlier up-regulation in the resistant snails but lower, slower protein expression in susceptible snails - was observed by Western blot analysis. Enzymatic analysis of the purified, recombinant BgPrx4 revealed the snail sequence to function as Prx but with an unusual ability to use both thioredoxin and glutathione as substrates.

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

During helminth infections, alternatively activated macrophages (AAMacs) are key to promoting Th2 responses and suppressing Th1-driven inflammatory pathology. Th2 cytokines IL-4 and/or IL-13 are believed to be important in the induction and activation of AAMacs. Using murine models for the helminth infections caused by Fasciola hepatica (Fh) and Schistosoma mansoni (Sm), we show that a secreted antioxidant, peroxiredoxin (Prx), induces alternative activation of macrophages. These activated, Ym1-expressing macrophages enhanced the secretion of IL-4, IL-5, and IL-13 from naive CD4(+) T cells. Administration of recombinant FhPrx and SmPrx to wild-type and IL-4(-/-) and IL-13(-/-) mice induced the production of AAMacs. In addition, Prx stimulated the expression of markers of AAMacs (particularly, Ym1) in vitro, and therefore can act independently of IL-4/IL-13 signaling. The immunomodulatory property of Prx is not due to its antioxidant activity, as an inactive recombinant variant with active site Cys residues replaced by Gly could also induce AAMacs and Th2 responses. Immunization of mice with recombinant Prx or passive transfer of anti-Prx antibodies prior to infection with Fh not only blocked the induction of AAMacs but also the development of parasite-specific Th2 responses. We propose that Prx activates macrophages as an initial step in the induction of Th2 responses by helminth parasites and is thereby a novel pathogen-associated molecular pattern.

Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.

Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50)s ranging from micromolar to the assay response limit ( approximately 25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases.

Despite the widespread use of chemotherapy and other control strategies over the past 50years, transmission rates for schistosomiasis have changed little. Regardless of the approach used, future control efforts will require a more complete understanding of fundamental parasite biology. Schistosomes undergo complex development involving an alteration of parasite generations within a mammalian and freshwater molluscan host in the completion of its lifecycle. Little is known about factors controlling schistosome development, but understanding these processes may facilitate the discovery of new control methods. Therefore, our goal in this study is to determine global developmentally regulated and stage-specific gene expression in Schistosoma mansoni using serial analysis of gene expression (SAGE). We present a preliminary analysis of genes expressed during development and sexual differentiation in the mammalian host and during early larval development in the snail host. A number of novel, differentially expressed genes have been identified, both within and between the different developmental stages found in the mammalian and snail hosts.

Schistosomiasis remains a largely neglected, global health problem. The morbid pathology of the disease stems from the host's inflammatory response to parasite eggs trapped in host tissues. Long term host/parasite survival is dependent upon the successful modulation of the acute pathological response, which is induced by egg antigens. In this study, using Multidimensional Protein Identification Technology, we identified the Schistosoma mansoni egg secretome consisting of 188 proteins. Notably we identified proteins involved in redox balance, molecular chaperoning and protein folding, development and signaling, scavenging and metabolic pathways, immune response modulation, and 32 novel, previously uncharacterized schistosome proteins. We localized a subset of previously characterized schistosome proteins identified in egg secretions in this study, to the surface of live S. mansoni eggs using the circumoval precipitin reaction. The identification of proteins actively secreted by live schistosome eggs provides important new information for understanding immune modulation and the pathology of schistosomiasis.

Background: Schistosomiasis--infection with helminth parasites in the genus Schistosoma, including S. mansoni--is a widespread, devastating tropical disease affecting more than 200 million people. No vaccine is available, and praziquantel, the only drug extensively utilized, is currently administered more than 100 million people yearly. Because praziquantel resistance may develop it is essential to identify novel drug targets. Our goal was to investigate the potential of a unique, selenium-containing parasite enzyme thioredoxin glutathione reductase (TGR) as a drug target., Methods and Findings: Using RNA interference we found that TGR is essential for parasite survival; after silencing of TGR expression, in vitro parasites died within 4 d. We also found that auranofin is an efficient inhibitor of pure TGR (Ki = 10 nM), able to kill parasites rapidly in culture at physiological concentrations (5 microM), and able to partially cure infected mice (worm burden reductions of ~60%). Furthermore, two previously used antischistosomal compounds inhibited TGR activity, suggesting that TGR is a key target during therapy with those compounds., Conclusions: Collectively, our results indicate that parasite TGR meets all the major criteria to be a key target for antischistosomal chemotherapy. To our knowledge this is the first validation of a Schistosoma drug target using a convergence of both genetic and biochemical approaches.

Schistosoma mansoni, a causative agent of schistosomiasis, resides in the hepatic portal circulation of their human host up to 30 years without being eliminated by the host immune attack. Production of an antioxidant "firewall," which would neutralize the oxidative assault generated by host immune defenses, is one proposed survival mechanism of the parasite. Schistosomes lack catalase, the main H2O2-neutralizing enzyme of many organisms, and their glutathione peroxidases are in the phospholipid class with poor reactivity toward H2O2. Evidence implicates peroxiredoxins (Prx) as providing the main enzymatic activity to reduce H2O2 in the parasite. Quantitative monitoring of Prx mRNAs during parasite life cycle indicated that Prx proteins are differentially expressed, with highest expression occurring in adult stages (oxidative resistant stages). Incubation of schistosomula with Prx1 double-stranded RNA knocked down total Prx enzymatic activity and resulted in lowered survival of cultured parasites compared with controls demonstrating that Prx are essential parasite proteins. These results represent the first report of lethal gene silencing in Schistosoma. Investigation of downstream effects of Prx silencing revealed an abrupt increase of lipid peroxides and the generation of several oxidized proteins. Using mass spectrometry, parasite albumin and actin were identified as the main oxidized proteins. Gene expression analysis showed that schistosome albumin was induced by oxidative stress. This study highlights Prx proteins as essential parasite proteins and potential new targets for anti-schistosome drug development and albumin as a novel, sacrificial oxidant scavenging protein in parasite redox regulation.

Pathology in schistosomiasis consists of granuloma formation around parasite eggs. There is considerable variation in the severity of disease in individuals with schistosomiasis, which may result from differential responses to egg antigens. The egg-induced immunopathology is mediated by CD4+ T helper cells sensitised to egg antigens. In this study, cellular responses to a 25-kDa fraction of egg proteins identified a novel T-cell antigen, SmEP25. The native SmEP25 elicited significant proliferative responses as well as gamma interferon (IFN-gamma), IL-2, IL-4, and IL-5 secretion in CD4+ cells from 8.5-week infected CBA and C57BL/6 mice. In C57BL/6 mice, proliferative responses to SmEP25 were relatively stronger than those directed against the major egg antigen Sm-p40, whereas in CBA mice the reverse was found. SmEP25 elicited stronger Th2 type response than Sm-p40 in both mouse strains. By comparison, recombinant SmEP25 elicited a smaller, Th1-polarised response, with significant IFN-gamma, low levels of IL-5 and essentially no IL-4. B-cell responses to SmEP25 coincided with the start of parasite egg production and SmEP25 protein was restricted to parasite eggs. The systematic identification of T-cell-sensitising egg components will lead to a better understanding of the processes involved in granuloma formation.

Peroxiredoxins are a large family of peroxidases that have important antioxidant and cell signaling functions. Genes encoding two novel 2-cysteine peroxiredoxin proteins were identified in the expressed sequence tag data base of the helminth parasite Schistosoma mansoni, a causative agent of schistosomiasis. The recombinant proteins showed peroxidase activity in vitro with a variety of hydroperoxides and used both the thioredoxin and the glutathione systems as electron donors. Steady-state kinetic analysis indicated that the new peroxiredoxins had saturable kinetics, whereas a previously identified schistosome peroxiredoxin was found to function with more typical unsaturable (ping-pong) kinetics. The catalytic efficiencies S. mansoni peroxiredoxins were similar to those for other peroxiredoxins studied (10(4)-10(5) m(-1) s(-1)). Mutagenesis of S. mansoni peroxiredoxins indicated that glutathione dependence and kinetic differences were conferred by the C-terminal alpha-helix forming 22 amino acids. This is the first report of 2-cysteine peroxiredoxins efficiently utilizing reducing equivalents from both the thioredoxin and glutathione systems. Studies to determine the resistance to oxidative inactivation, important in regulating cell signaling pathways, showed that S. mansoni possess both bacterial-like resistant and mammalian-like sensitive peroxiredoxins. The susceptibility to oxidative inactivation was conferred by the C-terminal tail containing a tyrosine-phenylalanine motif. S. mansoni is the first organism shown to possess both robust and sensitive peroxiredoxins. The ability of schistosome peroxiredoxins to use alternative electron donors, and their variable resistance to overoxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and its mammalian host.

The development and survival of the helminth parasite, Schistosoma mansoni, is dependent on its ability to interpret signals from its environment. Currently, little is known about signal transduction in schistosomes. Rho is a member of a super-family of small GTP-binding proteins. Rho is involved in a number of cell signalling pathways with effects on actin cytoskeleton organisation, gene transcription, cell cycle progression, and membrane trafficking. We have cloned an S. mansoni protein (Rho1) that has 71-75% identity and approximately 85% similarity with human Rho A, B, and C proteins. We have optimised expression of recombinant S. mansoni Rho1 protein in Escherichia coli by co-expression with rare tRNAs. Western blot analysis results showed expression of Rho1 protein in adult worm stages especially female worms. In vitro prenylation of recombinant S. mansoni Rho1 determined that, similar to Rho from other organisms, Rho1 is geranynlgeranylated but not farnesylated. A search of the gene database indicates that Rho GTPases exist as a small family in S. mansoni including orthologues of Rho, Cdc42, and Rac. These data suggest that S. mansoni Rho1 plays a role in signalling in adult worms, especially females.

The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated BOUDICCA: Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length. ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein, including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that of the gypsy/Ty3 retrotransposons. An additional ORF at the 3' end of the retrotransposon may encode an envelope protein. Phylogenetic comparison based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRn1 from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing approximately 8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode.

Adenylosuccinate lyase is an enzyme used in parasite nucleotide salvage pathways that cleaves adenylosuccinate into adenosine 5'-monophosphate and fumarate. A cDNA encoding adenylosuccinate lyase from the trematode parasite Schistosoma mansoni has been cloned for analysis. Sequencing of the cDNA revealed an open reading frame of 1454 nucleotides that codes for a protein with a predicted mass of about 54.5 kDa. Comparative analysis of the predicted protein sequence shows that S. mansoni adenylosuccinate lyase has a lot of similarity with human adenylosuccinate lyase. Genomic analysis using S. mansoni adenylosuccinate lyase-containing bacterial artificial chromosome (BAC) clones revealed a gene of approximately 19.4 kb consisting of eight exons and seven introns. Intron 6 was found to contain a novel 2.9 kb long terminal repeat retrotransposon with direct terminal repeats of 500 nucleotides. Fluorescence in situ hybridisation mapping localised S. mansoni adenylosuccinate lyase to the Z and W chromosomes. Analysis of S. mansoni adenylosuccinate lyase mRNA expression levels using real time reverse transcriptase (RT)-PCR showed that S. mansoni adenylosuccinate lyase is expressed at higher levels in the female worms than in the male worms and is expressed at different levels than other purine nucleotide salvage enzymes. Male homogenate showed a specific activity of 10.3 units/mg protein while the female showed a specific activity of 24.2 units/mg protein. These data indicate that S. mansoni adenylosuccinate lyase is an important parasite enzyme and should be examined as a potential chemotherapeutic target.

Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilising systems. Here, we report the identification and characterisation of a thioredoxin from Schistosoma mansoni. The predicted protein has similarity to previously characterised thioredoxins including conservation of the redox active site. Recombinant six-histidine tagged schistosome thioredoxin had insulin reduction activity and supported the enzymatic function of thioredoxin reductase and thioredoxin peroxidase. By Western blotting, all mammalian stages of the schistosome lifecycle expresses thioredoxin. Thioredoxin is present in egg secretory products and antibodies against the recombinant protein produce the circumoval precipitin reaction. This is the first identification of defined antigen producing this reaction. Furthermore, thioredoxin is a novel egg immunogen as it elicits an antibody response in schistosome-infected mice. The most significant IgG production against thioredoxin occurs after parasite oviposition commences. These observations suggest that thioredoxin participates in processes vital to the parasite and may facilitate the passage and survival of eggs across inflamed host tissues.

Schistosoma mansoni, a causative agent of schistosomiasis, is a major cause of human morbidity in tropical countries. Adult schistosomes, which reside in the hepatic portal system, are exposed to reactive oxygen compounds through respiration and as a result of the host immune response. To minimize oxidative stress schistosomes must possess adequate mechanisms of detoxification. Major detoxification systems rely on reducing equivalents from the disulfide oxidoreductases glutathione and thioredoxin. Therefore, maintenance of adequate levels of these thiols in a reduced form is critical. Here we show that S. mansoni possess an unusual thiol redox system centered on thioredoxin glutathione reductase. This enzyme represents an unusual fusion of a pyridine nucleotide disulfide oxidoreductase with a redox active glutaredoxin extension. Furthermore, we predict that this is a selenocysteine protein. Immunoprecipitation, western blot and inhibitor studies show that this protein has thioredoxin reductase, glutathione reductase, and glutaredoxin activities. Most importantly, we show that thioredoxin glutathione reductase appears to be the major, if not the sole enzyme for these activities in adult worms, completely replacing thioredoxin reductase and glutathione reductase. This is the first example of an organism with a redox system based exclusively on thioredoxin glutathione reductase.

The ability of Schistosoma mansoni to escape oxidative damage from immune system-generated reactive oxygen intermediates has been extensively documented. The limiting step in the parasite's detoxification process appears to be at the level of hydrogen peroxide neutralization. In the present study, the possible role of a novel class of antioxidant enzymes, thioredoxin peroxidase (TPx), in hydrogen peroxide neutralization by schistosomes was investigated. An expressed sequence tag was characterized from the Schistosoma Genome Initiative with high similarity to TPx from other organisms. The gene encodes a polypeptide containing 2 conserved active-site cysteines and flanking amino acids, and 60-70% identity with previously characterized TPx proteins. Recombinant schistosome TPx was enzymatically active and found to have thioredoxin-dependent hydrogen peroxide reducing activity of 4500 nmol hydrogen peroxide/min/mg protein. Native TPx activity was determined to be 48.1 nmol hydrogen peroxide/min/mg protein in adult worm homogenates compared with 46.9 for glutathione peroxidase. TPx activity was precipitated from adult worm homogenates with antibodies prepared against the recombinant protein. Western blotting with antibodies made against recombinant protein showed that TPx was expressed in both male and female adult worms. This is the first demonstration of a TPx activity in schistosomes and our results suggest that TPx plays a significant role in schistosome-host interactions.

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen., (Copyright 2000 Academic Press.)

Antioxidant enzymes are thought to play a crucial role in the survival of the parasite, Schistosoma mansoni, during its migration through the tissues of the definitive host. We recently cloned the cDNA encoding one such enzyme, glutathione peroxidase (Gpx). In order to elucidate the regulation of expression of this gene, we describe the cloning and characterization of a Gpx gene of S. mansoni. An initial screen of a lambda EMBL4 genomic library using the corresponding cDNA sequence as a probe yielded 14 positive clones, two of which have so far been analyzed in detail. The complete Gpx gene contains five introns, four of which, located at the 5' end, are extremely short (30-51 bp) and the last of which is approximately 6 kb long. We present the sequence of the gene including 73 bp at the 5' end, the complete sequence to 137 bp downstream from the penultimate exon, 164 bp upstream and 131 bp downstream from the last 3' exon. The potential mRNA cap site is situated 219 bp upstream from the ATG start codon. All intron/exon junctions correspond to the conventional eukaryotic splice signal. Analysis of the 5' flanking region revealed the presence of a potential TATA box at--26 bp from the cap site, but no CAAT-like element is present. Southern blot analysis showed a unique Gpx gene organisation in the S. mansoni genome.