Characterisation of major vault protein during the life cycle of the human parasite Schistosoma mansoni

Vaults are ribonucleoproteins (13 MDa) highly conserved among lower and higher eukaryotes. Their association produces a complex composed of three proteins named Major Vault Protein (MVP), vault (PolyADP-ribose) polymerase (VPARP) and Telomerase-associated protein (TEP1), plus a small untranslated RNA. The exact function of this complex is unknown, although the biological role of vaults has been associated with multidrug resistance phenotypes and signal transduction pathways. Genomic analysis showed that model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, do not possess genes encoding vaults. However, we have found that vault-related genes are present in the Schistosoma mansoni genome. These observations raised questions on the involvement of vaults in mechanisms of adaptation of the parasite in its mammalian host. Therefore, molecular characterisation of the putative Major Vault Protein performed using bioinformatics tools showed that this vault component is highly conserved in S. mansoni. The MVP expression level was quantified by qRT-PCR using total RNA from susceptible (LE) and resistant (LE-PZQ) adult worm lineages, cercariae and mechanically transformed schistosomula (MTS) cultured for 3.5, 24, 48 and 72 h in vitro. Our results suggest a stage-specific expression in all developmental stages analysed. Western blotting has shown up-regulation of SmMVP in the MTS-3.5, 72 h and resistant adult worms, and similar levels in all other stages. Furthermore, SmMVP was found differentially expressed in adult males and females from the susceptible lineage. Further studies should clarify whether SmMVP is somehow linked to drug resistance in S. mansoni.     

Double-Stranded RNA Uptake through Topical Application,Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in Diaphorina citri

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P₄₅₀ genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P₄₅₀ proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.     

Refolding of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiasis

Schistosomiasis is an important parasitic disease, with about 240 million people infected worldwide. Humans and animals can be infected, imposing an enormous social and economic burden. The only drug available for chemotherapy, praziquantel, does not control reinfections, and an efficient vaccine for prophylaxis is still missing. However, the tegumental protein Sm29 of Schistosoma mansoni was shown to be a promising antigen to compose an anti-schistosomiasis vaccine. Though, recombinant Sm29 is expressed in Escherichia coli as insoluble inclusion bodies requiring an efficient process of refolding, thus, hampering its production in large scale. We present in this work studies to refold the recombinant Sm29 using high hydrostatic pressure, a mild condition to dissociate aggregated proteins, leading to refolding on a soluble conformation. Our studies resulted in high yield of rSm29 (73%) as a stably soluble and structured protein. The refolded antigen presented protective effect against S. mansoni development in immunized mice. We concluded that the refolding process by application of high hydrostatic pressure succeeded, and the procedure can be scaled-up, allowing industrial production of Sm29.     

Binding of a Cyano- and Fluoro-containing Drug Bicalutamide to Cytochrome P450 46A1: UNUSUAL FEATURES AND SPECTRAL RESPONSE*

Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.     

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium

BackgroundAnthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay.Methodology/Principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3–6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug.ConclusionWith this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).     

Ameliorative effects of Schisandrin B on Schistosoma mansoni-induced hepatic fibrosis in vivo

Schistosomiasis is second only to malaria as the most devastating parasitic disease in the world. It is caused by the helminths Schistosoma mansoni (S. mansoni), S. haematobium, or S. japonicum. Typically, patients with schistosomiasis suffer from symptoms of liver fibrosis and hepatosplenomegaly. Currently, patients were treated with praziquantel. Although praziquantel effectively kills the worm, it cannot prevent re-infection or resolve liver fibrosis. Also, current treatment options are not ample to completely cure liver fibrosis and splenic damages. Moreover, resistance of praziquantel has been reported in vivo and in vitro studies. Therefore, finding new effective treatment agents is urgently needed. Schisandrin B (Sch B) of Schisandra chinensis has been shown to protect against different liver injuries including fatty liver disease, hepatotoxicity, fibrosis, and hepatoma. We herein investigate the potential of using Sch B to treat S. mansoni-induced liver fibrosis. Results from the present study demonstrate that Sch B is beneficial in treating S. mansoni-induced liver fibrosis and splenic damages, through inhibition of inflammasome activation and apoptosis; and aside from that regulates host immune responses. Besides, Sch B treatment damages male adult worm in the mice, consequently helps to reduce egg production and lessen the parasite burden.     

Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min⁻¹, respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.     

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.     

Schistosome Syntenin Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund''s adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30–37% reduction of worm burden, 38–43% reduction in the number, and 35–37% reduction in the area, of liver granulomas.

Conclusions/Significance

Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.     

Sm10.3, a Member of the Micro-Exon Gene 4 (MEG-4) Family,Induces Erythrocyte Agglutination In Vitro and Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis.

Conclusions/Significance

Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.     

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus

The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.     

Mefloquine—An Aminoalcohol with Promising Antischistosomal Properties in Mice

Background

The treatment and control of schistosomiasis, an often neglected tropical disease that exacerbates poverty, depends on a single drug, praziquantel. The large-scale use of praziquantel might select for drug-resistant parasites, hence there is a need to develop new antischistosomal compounds. Here, we report that the antimalarial drug mefloquine possesses promising antischistosomal properties in mice.

Methodology/Principal Findings

A single dose of mefloquine (200 or 400 mg/kg) administered orally to mice infected with adult Schistosoma mansoni or adult S. japonicum resulted in high or complete total and female worm burden reductions (72.3%–100%). Importantly, high worm burden reductions were also observed for young developing stages of S. mansoni and S. japonicum harbored in the mouse. Both mefloquine erythro-enantiomers resulted in high and comparable total and female worm burden reductions when given to mice with either a sub-patent or patent S. mansoni infection.

Conclusions/Significance

Our findings hold promise for the development of a novel antischistosomal drug based on an aminoalcohol functionality. Further in vitro and in vivo studies have been launched to elucidate the possible mechanism of action and to study the effect of mefloquine on S. haematobium and other trematodes. It will be interesting to investigate whether mefloquine, which is widely and effectively used for the treatment of malaria, has an impact on schistosomiasis in areas where both malaria and schistosomiasis co-exist.     

Validation of a human-serum-based in vitro growth method for drug screening on juvenile development stages of Schistosoma mansoni

BackgroundSchistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.Methodology/Principal findingsWe compared inter–batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC₅₀: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC₅₀: 4.43 μM in vitro, 0.48 μM in vivo).ConclusionA large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts.     

Worm Proteins of Schistosoma mansoni Reduce the Severity of Experimental Chronic Colitis in Mice by Suppressing Colonic Proinflammatory Immune Responses

Although helminthic therapy as a possible new option to treat inflammatory bowel disease is a well-established concept by now, the search for immunomodulatory helminth-derived compounds and their mechanisms of action is still ongoing. We investigated the therapeutic potential and the underlying immunological mechanisms of Schistosoma mansoni soluble worm proteins (SmSWP) in an adoptive T cell transfer mouse model of chronic colitis. Both a curative and a preventive treatment protocol were included in this study. The curative administration of SmSWP (started when colitis was established), resulted in a significant improvement of the clinical disease score, colonoscopy, macroscopic and microscopic inflammation score, colon length and myeloperoxidase activity. The therapeutic potential of the preventive SmSWP treatment (started before colitis was established), was less pronounced compared with the curative SmSWP treatment but still resulted in an improved clinical disease score, body weight loss, colon length and microscopic inflammation score. Both the curative and preventive SmSWP treatment downregulated the mRNA expression of the proinflammatory cytokines IFN-γ and IL-17A and upregulated the mRNA expression of the anti-inflammatory cytokine IL-4 in the colon at the end of the experiment. This colonic immunomodulatory effect of SmSWP could not be confirmed at the protein level. Moreover, the effect of SmSWP appeared to be a local colonic phenomenon, since the flow cytometric T cell characterization of the mesenteric lymph nodes and the cytokine measurements in the serum did not reveal any effect of SmSWP treatment. In conclusion, SmSWP treatment reduced the severity of colitis in the adoptive transfer mouse model via the suppression of proinflammatory cytokines and the induction of an anti-inflammatory response in the colon.     

Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cytochrome P450 BM3

Cytochrome P450 enzymes (P450s or CYPs) are good candidates for biocatalysis in the production of fine chemicals, including pharmaceuticals. Despite the potential use of mammalian P450s in various fields of biotechnology, these enzymes are not suitable as biocatalysts due to their low stability, low catalytic activity, and limited availability. Recently, wild-type and mutant forms of bacterial P450 BM3 (CYP102A1) from Bacillus megaterium have been found to metabolize various. It has therefore been suggested that CYP102A1 may be used to generate the metabolites of drugs and drug candidates. In this report, we show that the oxidation reactions of typical human CYP1A2 substrates (phenacetin, ethoxyresorufin, and methoxyresorufin) are catalyzed by both wild-type and mutant forms of CYP102A1. In the case of phenacetin, CYP102A1 enzymes show only O-deethylation product, even though two major products are produced as a result of O-deethylation and 3-hydroxylation reactions by human CYP1A2. Formation of the metabolites was confirmed by HPLC analysis and LC–MS to compare the metabolites with the actual biological metabolites produced by human CYP1A2. The results demonstrate that CYP102A1 mutants can be used for cost-effective and scalable production of human CYP1A2 drug metabolites. Our computational findings suggest that a conformational change in the cavity size of the active sites of the mutants is dependent on activity change. The modeling results further suggest that the activity change results from the movement of several specific residues in the active sites of the mutants.