Parasitic Flatworm Schistosoma Mansoni Research Articles

Dual Strategy to Design New Agents Targeting Schistosoma mansoni: Advancing Phenotypic and SmCB1 Inhibitors for Improved Efficacy.

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 μM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 μM, 68% at 1 μM, 35% at 0.1 μM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

Convergent evolution of a genotoxic stress response in a parasite-specific p53 homolog

P53 is a widely studied tumor suppressor that plays important roles in cell-cycle regulation, cell death, and DNA damage repair. P53 is found throughout metazoans, even in invertebrates that do not develop malignancies. The prevailing theory for why these invertebrates possess a tumor suppressor is that P53 originally evolved to protect the germline of early metazoans from genotoxic stress such as ultraviolet radiation. This theory is largely based upon functional data from only three invertebrates, omitting important groups of animals including flatworms. Previous studies in the freshwater planarian flatworm Schmidtea mediterranea suggested that flatworm P53 plays an important role in stem cell maintenance and skin production, but these studies did not directly test for any tumor suppressor functions. To better understand the function of P53 homologs across diverse flatworms, we examined the function of two different P53 homologs in the parasitic flatworm Schistosoma mansoni. The first P53 homolog (p53-1) is orthologous to S. mediterranea P53(Smed-p53) and human TP53 and regulates flatworm stem cell maintenance and skin production. The second P53 homolog (p53-2) is a parasite-specific paralog that is conserved across parasitic flatworms and is required for the normal response to genotoxic stress in S. mansoni. We then found that Smed-p53 does not seem to play any role in the planarian response to genotoxic stress. The existence of this parasite-specific paralog that bears a tumor suppressor-like function in parasitic flatworms implies that the ability to respond to genotoxic stress in parasitic flatworms may have arisen from convergent evolution.

Structural and enzymatic insights into species-specific resistance to schistosome parasite drug therapy

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Schistosoma mansoni. Of the three main human schistosome species, only S. mansoni is sensitive to oxamniquine therapy despite the presence of SULT orthologs in Schistosoma hematobium and Schistosoma japonicum The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of S. hematobium and S. japonicum SULTs, including S. hematobium SULT in complex with oxamniquine. We also examined the activity of the three enzymes in vitro; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy.

The Planorbid Snail Biomphalaria glabrata Expresses a Hemocyanin-Like Sequence in the Albumen Gland.

The parasitic flatworm Schistosoma mansoni, causative agent of human intestinal schistosomiasis in South America, relies importantly on the freshwater snail Biomphalaria glabrata as intermediate host to achieve development of cercariae that infect humans. The recommendation from the World Health Organization (WHO) to integrate snail control in efforts to counter schistosomiasis transmission provides impetus for in depth study of B. glabrata biology. Our analysis indicates that two distinct hemocyanin-like genes (hcl-1 and hcl-2) are present in B. glabrata, a snail that uses hemoglobin for oxygen transport. Characterization of BAC clones yielded the full length hcl-1 gene, which is comprised of three functional unit (FU) domains at the amino acid level. Database searches and in silico analyses identified the second hcl gene (hcl-2), composed of six FU domains. Both genes are unusual for lacking canonical residues and having fewer FU domains than typical molluscan hemocyanins that contain 7–8 FUs. Reverse transcription PCR demonstrated that Hcl-1 is expressed in a manner that correlates with reproductive maturity in the albumen gland (AG), an immune- and reproduction-relevant organ. Immune cross-reactivity with anti-keyhole limpet hemocyanin (α-KLH) antiserum and tandem-mass spectrometry validated the presence of Hcl-1 protein in the AG and egg mass fluid (EMF). The evolutionary conservation of hemocyanin-like sequences in B. glabrata in the presence of the oxygen carrier hemoglobin, combined with our results, suggest that the Hcl-1protein has a functional role in general and/or reproductive biology. Further investigations are needed to explore Hcl-1 as a potential target for snail control.

Comparative pharmacology of flatworm and roundworm glutamate-gated chloride channels: Implications for potential anthelmintics

Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0±0.1mM, 2.4±0.4mM, 3.6±0.7mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484±85μM) and least potently by menthol (IC50>3mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

Molecular Basis for the Antiparasitic Activity of a Mercaptoacetamide Derivative That Inhibits Histone Deacetylase 8 (HDAC8) from the Human Pathogen Schistosoma mansoni

Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion and the essential catalytic tyrosine (Y341) residue via its mercaptoacetamide warhead. To our knowledge, this is the first complex crystal structure of any HDAC inhibited by a mercaptoacetamide inhibitor, and therefore, this finding offers a rationale for further improvement. Finally, an ester prodrug of the thiol HDAC inhibitor exhibited antiparasitic activity on cultured schistosomes in a dose-dependent manner.

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.

BackgroundThe 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 FindingsIn 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/SignificanceOur 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.

Fatty Acid Oxidation Is Essential for Egg Production by the Parasitic Flatworm Schistosoma mansoni

Schistosomes, parasitic flatworms that cause the neglected tropical disease schistosomiasis, have been considered to have an entirely carbohydrate based metabolism, with glycolysis playing a dominant role in the adult parasites. However, we have discovered a close link between mitochondrial oxygen consumption by female schistosomes and their ability to produce eggs. We show that oxygen consumption rates (OCR) and egg production are significantly diminished by pharmacologic inhibition of carnitine palmitoyl transferase 1 (CPT1), which catalyzes a rate limiting step in fatty acid β-oxidation (FAO) and by genetic loss of function of acyl CoA synthetase, which complexes with CPT1 and activates long chain FA for use in FAO, and of acyl CoA dehydrogenase, which catalyzes the first step in FAO within mitochondria. Declines in OCR and egg production correlate with changes in a network of lipid droplets within cells in a specialized reproductive organ, the vitellarium. Our data point to the importance of regulated lipid stores and FAO for the compartmentalized process of egg production in schistosomes.

Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3′end, suggesting the transcript is full-length. The cloned cDNA was subsequently expressed in bacteria and shown to encode a functional glutamate-binding protein. Other studies, using a specific peptide antibody, determined that SmGBP exists in two forms, a monomer of the expected size and a stable but non-covalent dimer. The monomer and dimer are both present in the membrane fraction of S. mansoni and are resistant to extraction with high-salt, alkaline pH and urea, suggesting SmGBP is either an integral membrane protein or a peripheral protein that is tightly associated with the membrane. Surface biotinylation experiments combined with western blot analyses and confocal immunolocalization revealed that SmGBP localized to the surface membranes of adult male schistosomes, especially the dorsal tubercles. In contrast, we detected little or no expression of SmGBP either in the females or larval stages. A comparative quantitative PCR analysis confirmed that the level of SmGBP expression is several-fold higher in male worms than cercariae, and it is barely detectable in adult females. Together, the results identify SmGBP as a new type of schistosome glutamate receptor that is both gender- and stage-specific. The high-level expression of this protein in the male tubercles suggests a possible role in host-parasite interaction.

Cloning and Functional Characterization of Two Calmodulin Genes During Larval Development in the Parasitic Flatworm Schistosoma mansoni

Schistosomiasis is endemic in over 70 countries, in which more than 200 million people are infected with the various schistosome species. Understanding the physiological processes underlying key developmental events could be useful in developing novel chemotherapeutic reagents or infection intervention strategies. Calmodulin is a small, calcium-sensing protein found in all eukaryotes and, although the protein has been previously identified in various Schistosoma mansoni stages and implicated in egg hatching and miracidia transformation, few molecular and functional data are available for this essential protein. Herein, we report the molecular cloning, expression, and functional characterization of calmodulin in the miracidia and primary sporocyst stages of S. mansoni. Two transcripts, SmCaM1 and SmCaM2, were cloned and sequenced, and a recombinant SmCaM1 protein was expressed in Escherichia coli and used to generate anti-CaM antibodies. The 2 protein sequences were highly conserved when compared to other model organisms. The alignment of the predicted proteins of both SmCaM1 and SmCaM2 exhibited 99% identity to each other and 97-98% identity with mammalian calmodulins. Analysis of steady-state transcript abundance indicate that the 2 calmodulin transcripts differ in their stage-associated expression patterns, although the CaM protein isotype appears to be constitutively expressed during early larval development. Application of RNAi to larval parasites results in a "stunted growth" phenotype in sporocysts with 30 and 35% reduction in transcript abundance for SmCaM1 and SmCaM2, respectively, and a corresponding 35% reduction in protein level after incubation in double-stranded RNA. Differential expression of CaM transcripts during early larval development and a growth defect-inducing effect associated with partial transcript and protein inhibition as a result of RNAi suggest a potentially important role of calmodulin during early larval development.

Crystallization and X-ray analysis of theSchistosoma mansoniguanidino kinase

The 716-amino-acid guanidino kinase from the parasitic flatworm Schistosoma mansoni results from the fusion of two guanidino kinase subunits. Crystals of this 80 kDa protein have been obtained in the monoclinic space group P2(1), with unit-cell parameters a = 52.7, b = 122.1, c = 63.2 A, beta = 108.5 degrees . Synchrotron data were collected to 2.8 A resolution on ESRF beamline ID29. The structure was solved by the molecular-replacement method, using the 357-amino-acid structure of the arginine kinase from Trypanosoma cruzi as the search model.

A bone morphogenetic protein homologue in the parasitic flatworm, Schistosoma mansoni

Members of the bone morphogenetic protein (BMP) subfamily of cytokines control many aspects of metazoan development including patterning and organogenesis. Despite the recognition that schistosomes possess key components of a BMP signaling pathway, a BMP-like ligand in the parasitic flatworm Schistosoma mansoni remained elusive. Here, we describe the cloning and characterisation of an S. mansoni BMP ( SmBMP). SmBMP is most closely related to BMP homologues from the free-living flatworms Schmidtea mediterranea and Dugesia japonica, with 51% and 47% identity at the amino acid level, respectively. Based on reverse transcription-PCR, SmBMP is expressed throughout the mammalian life-cycle of the parasite in both male and female schistosomes. In support of these results, antibodies to SmBMP successfully immunoprecipitated the protein in adult male and female antigen preparations with more protein detected in male parasites. Immunofluorescent studies localised SmBMP to the protonephridia of adult parasites, and SmBMP was identified in the excretory/secretory products of adult male parasites via immunoprecipitation. With the previous description of a TGF-β subfamily homologue in S. mansoni, ligands representing both arms of the TGF-β superfamily have now been described in this trematode.

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Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

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Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 μM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

Do mice genetically selected for resistance to oral tolerance provide selective advantage for Schistosoma mansoni infection?

A highly evolved relationship exists between the parasitic flatworm Schistosoma mansoni and its vertebrate hosts that include the use of host immune signals by parasites. The S. mansoni infection was studied in two strains of mice genetically selected, over 18 generations of assortative mating, for extreme phenotypes of susceptibility (TS) and resistance (TR) to immunological tolerance. The objective was to observe whether the different host genetic backgrounds affected the outcome of experimental schistosomiasis. Fecal egg excretion, tissue egg count, worm recovery, and adult worm morphology and morphometry were monitored throughout the period of infection. TR mice presented total fecal egg excretion and thickness of tegument in adult male worms significantly higher than TS mice. Therefore, the comparative analysis of mice with extreme phenotypes of immunological response turns out to be useful in host–parasite relationship studies. Our results suggest that the TR mouse immunological profile provides a more favorable environment for the development of S. mansoni.