Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

The function of twister ribozyme variants in non-LTR retrotransposition in Schistosoma mansoni.

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5' end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5' end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.

CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signalling and drug detoxification and are essential for parasite survival.

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.

Binding Free Energy (BFE) Calculations and Quantitative Structure-Activity Relationship (QSAR) Analysis of Schistosoma mansoni Histone Deacetylase 8 (smHDAC8) Inhibitors.

Histone-modifying proteins have been identified as promising targets to treat several diseases including cancer and parasitic ailments. In silico methods have been incorporated within a variety of drug discovery programs to facilitate the identification and development of novel lead compounds. In this study, we explore the binding modes of a series of benzhydroxamates derivatives developed as histone deacetylase inhibitors of Schistosoma mansoni histone deacetylase (smHDAC) using molecular docking and binding free energy (BFE) calculations. The developed docking protocol was able to correctly reproduce the experimentally established binding modes of resolved smHDAC8-inhibitor complexes. However, as has been reported in former studies, the obtained docking scores weakly correlate with the experimentally determined activity of the studied inhibitors. Thus, the obtained docking poses were refined and rescored using the Amber software. From the computed protein-inhibitor BFE, different quantitative structure-activity relationship (QSAR) models could be developed and validated using several cross-validation techniques. Some of the generated QSAR models with good correlation could explain up to ~73% variance in activity within the studied training set molecules. The best performing models were subsequently tested on an external test set of newly designed and synthesized analogs. In vitro testing showed a good correlation between the predicted and experimentally observed IC50 values. Thus, the generated models can be considered as interesting tools for the identification of novel smHDAC8 inhibitors.

A metalloprotease produced by larval Schistosoma mansoni facilitates infection establishment and maintenance in the snail host by interfering with immune cell function.

Metalloproteases (MPs) have demonstrated roles in immune modulation. In some cases, these enzymes are produced by parasites to influence host immune responses such that parasite infection is facilitated. One of the best examples of parasite-mediated immune modulation is the matrix metalloprotease (MMP) leishmanolysin (Gp63), which is produced by species of the genus Leishmania to evade killing by host macrophages. Leishmanolysin-like proteins appear to be quite common in many invertebrates, however our understanding of the functions of these non-leishmania enzymes is limited. Numerous proteomic and transcriptomic screens of schistosomes, at all life cycle stages of the parasite, have identified leishmanolysin-like MPs as being present in abundance; with the highest levels being found during the intramolluscan larval stages and being produced by cercaria. This study aims to functionally characterize a Schistosoma mansoni variant of leishmanolysin that most resembles the enzyme produced by Leishmania, termed SmLeish. We demonstrate that SmLeish is an important component of S. mansoni excretory/secretory (ES) products and is produced by the sporocyst during infection. The presence of SmLeish interferes with the migration of Biomphalaria glabrata haemocytes, and causes them to present a phenotype that is less capable of sporocyst encapsulation. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to susceptible B. glabrata reduces miracidia penetration success, causes a delay in reaching patent infection, and lowers cercaria output from infected snails.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo.

Praziquantel (PZQ) is the sole drug used to treat schistosomiasis, and the probability of developing resistance is growing the longer it is relied upon, justifying the search for alternatives. Phosphodiesterases (PDEs), particularly the PDE4 family, have attracted considerable attention as drug targets, including in Schistosoma mansoni, and especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development.

Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools.

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide-binding domain. To get functional information on SMA1, we produced it in an active form in Saccharomyces cerevisiae, as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase, whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite.

In Vitro and in Vivo Antischistosomal Activities of Chalcones.

In this study, we evaluated the in vitro and in vivo schistosomicidal activities of chalcones against Schistosoma mansoni worms. In vitro assays revealed that chalcones 1 and 3 were the most active compounds, without affecting significantly mammalian cells. Confocal laser scanning microscopy and scanning electron microscopy studies revealed reduction on the numbers of tubercles and morphological alterations in the tegument of S. mansoni worms after in vitro incubation with chalcones 1 and 3. In a mouse model of schistosomiasis, the oral treatment (400 mg/kg) with chalcone 1 or 3 significantly caused a total worm burden reduction in mice. Chalcone 1 showed significant inhibition of the S. mansoni ATP diphosphohydrolase activity, which was corroborated by molecular docking studies. The results suggested that chalcones could be explored as lead compounds with antischistosomal properties.

A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing.

A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 values ranging from 4.4-20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure-activity relationship.

The natural compound 7-epiclusianone inhibits superoxide dismutase activity in Schistosoma mansoni.

Schistosomiasis - caused by trematodes from the genus Schistosoma - affects more than 200 million people worldwide. Growing resistance to therapy with praziquantel (PZQ) has encouraged the search for novel treatments against this neglected disease. The compound 7-epiclusianone (7-epi) - isolated from 'bacupari' (the fruit of the Gracinia brasiliensis tree) - has promising activity against Schistosoma mansoni in vitro, damaging the parasite's tegument. However, the target and mechanism of action of 7-epi have not been identified. Here, we examined the possibility that 7-epi harms the tegument by inhibiting parasite superoxide dismutase (SOD), which protects the tegument from damage by reactive oxygen species produced by host immune cells. Molecular docking analysis in silico suggested strong interactions between 7-epi and S. mansoni cytosolic superoxide dismutase (SmCtSOD) at allosteric cavities. Schistosoma mansoni couples were cultivated ex vivo with 12.44-198.96 µm 7-epi for 24 h, and then parasite extracts were tested for lipid peroxidation (as a surrogate for oxidative stress), and SOD activity and expression. Lipid peroxidation levels increased after incubation with concentrations ≥99.48 µm 7-epi, and this compound reduced SOD activity at concentrations ≥24.87 µm. However, contact with 7-epi did not alter SOD expression, by quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that the inhibition of SmCtSOD is partly responsible for the tegument detachment observed after incubation with 7-epi, but is not the only cause of the antiparasitic action of this compound in vitro.

Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase.

Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite's ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.

Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) deaminase.

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.

The helminth T2 RNase ω1 promotes metabolic homeostasis in an IL-33- and group 2 innate lymphoid cell-dependent mechanism.

Induction of a type 2 cellular response in the white adipose tissue leads to weight loss and improves glucose homeostasis in obese animals. Injection of obese mice with recombinant helminth-derived Schistosoma mansoni egg-derived ω1 (ω1), a potent inducer of type 2 activation, improves metabolic status involving a mechanism reliant upon release of the type 2 initiator cytokine IL-33. IL-33 initiates the accumulation of group 2 innate lymphoid cells (ILC2s), eosinophils, and alternatively activated macrophages in the adipose tissue. IL-33 release from cells in the adipose tissue is mediated by the RNase activity of ω1; however, the ability of ω1 to improve metabolic status is reliant upon effective binding of ω1 to CD206. We demonstrate a novel mechanism for RNase-mediated release of IL-33 inducing ILC2-dependent improvements in the metabolic status of obese animals.