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[This corrects the article DOI: 10.3389/fmicb.2023.1079855.].
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Introduction: The human blood fluke parasite Schistosoma mansoni relies on diverse mechanisms to adapt to its diverse environments and hosts. Epigenetic mechanisms play a central role in gene expression regulation, culminating in such adaptations. Protein arginine methyltransferases (PRMTs) promote posttranslational modifications, modulating the function of histones and non-histone targets. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is one of the S. mansoni proteins with the PRMT core domain. Methods: We carried out in silico analyses to verify the expression of SmPRMTs in public datasets from different infection stages, single-sex versus mixed-worms, and cell types. The SmCARM1 function was evaluated by RNA interference. Gene expression levels were assessed, and phenotypic alterations were analyzed in vitro, in vivo, and ex vivo. Results: The scRNAseq data showed that SmPRMTs expression is not enriched in any cell cluster in adult worms or schistosomula, except for Smcarm1 expression which is enriched in clusters of ambiguous cells and Smprmt1 in NDF+ neurons and stem/germinal cells from schistosomula. Smprmt1 is also enriched in S1 and late female germ cells from adult worms. After dsRNA exposure in vitro, we observed a Smcarm1 knockdown in schistosomula and adult worms, 83 and 69%, respectively. Smcarm1-knockdown resulted in reduced oviposition and no significant changes in the schistosomula or adult worm phenotypes. In vivo analysis after murine infection with Smcarm1 knocked-down schistosomula, showed no significant change in the number of worms recovered from mice, however, a significant reduction in the number of eggs recovered was detected. The ex vivo worms presented a significant decrease in the ovary area with a lower degree of cell differentiation, vitelline glands cell disorganization, and a decrease in the testicular lobe area. The worm tegument presented a lower number of tubercles, and the ventral sucker of the parasites presented a damaged tegument and points of detachment from the parasite body. Discussion: This work brings the first functional characterization of SmCARM1 shedding light on its roles in S. mansoni biology and its potential as a drug target. Additional studies are necessary to investigate whether the reported effects of Smcarm1 knockdown are a consequence of the SmCARM1-mediated methylation of histone tails involved in DNA packaging or other non-histone proteins.
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Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.
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Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/ultraestructura , Esquistosomiasis mansoni/tratamiento farmacológico , Animales , Antihelmínticos/farmacología , Resistencia a Medicamentos , Microscopía Electrónica de Rastreo , Oviposición/efectos de los fármacos , Praziquantel/farmacología , Esquistosomiasis mansoni/parasitología , Esquistosomiasis mansoni/patologíaRESUMEN
Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.
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BACKGROUND: The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. CONCLUSIONS/SIGNIFICANCE: The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents.
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Replicación del ADN/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Indazoles/farmacología , Oviposición/efectos de los fármacos , Piridonas/farmacología , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/fisiología , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Locomoción/efectos de los fármacos , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Microscopía Electrónica de Rastreo , ARN no Traducido/metabolismo , Schistosoma mansoni/enzimología , Schistosoma mansoni/ultraestructura , Análisis de SupervivenciaAsunto(s)
Antihelmínticos/farmacología , Descubrimiento de Drogas , Genómica , Schistosoma/efectos de los fármacos , Schistosoma/genética , Esquistosomiasis/tratamiento farmacológico , Esquistosomiasis/parasitología , Animales , Descubrimiento de Drogas/métodos , Genómica/métodos , Humanos , Terapia Molecular Dirigida/métodos , Schistosoma/fisiologíaRESUMEN
Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.
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Antihelmínticos/uso terapéutico , Proteínas del Huevo/genética , Epigénesis Genética/fisiología , Oviparidad/genética , Schistosoma mansoni/fisiología , Esquistosomiasis/prevención & control , Esquistosomiasis/transmisión , Animales , Proteínas del Huevo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células HEK293 , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Masculino , Terapia Molecular Dirigida/métodos , Schistosoma mansoni/genética , Schistosoma mansoni/crecimiento & desarrolloRESUMEN
Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel ß-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.
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Proteínas del Helminto/química , Modelos Moleculares , Factores de Transcripción/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Schistosoma mansoni is a human endoparasite with a complex life cycle that also infects an invertebrate mollusk intermediate host and exhibits many diverse phenotypes. Its complexity is reflected in a large genome and different transcriptome profiles specific to each life cycle stage. Epigenetic regulation of gene expression such as the post-translational modification of histones has a significant impact on phenotypes, and this information storage function resides primarily at histone tails, which results in a varied histone code. Evidence of transcription of the different histone families at all life stages of the parasite was detected by a survey of transcriptome databases; manual curation of each gene prediction at the genome sequence level showed errors in the coding sequences of three of them. The biogenesis of histones is coupled to DNA replication, and a detailed in silico analysis of the specialized machinery of histone mRNA processing in the S. mansoni genome reveals that it is as conserved as in other eukaryotes, consisting in transcription factors and stem-loop binding proteins which recognize the stem loop structure at the histone mRNA 3'UTR. Histone modifying enzymes (HMEs) such as histone acetyltransferases, methyltransferases and deacetylases (HDACs) have been described in S. mansoni, and their potential as new therapeutic targets was evidenced with the apoptotic phenotype that resulted from HDAC inhibition. However, the overall regulation of transcription coupled with gene expression profiles correlated to histone modifications has not yet been characterized. Besides the interaction of HMEs with histones, many factors involved in cellular processes are known to bind to histones, and were identified here by an in silico analysis of the S. mansoni genome. Knowledge of the histone families opens up perspectives for further studies that will lead to a better identification of their post-translational modifications, their gene regulation and to the possible characterization of HMEs as targets for the development of new drugs.
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Cromatina/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Schistosoma mansoni/fisiología , Transcripción Genética , Animales , Epigénesis Genética , Procesamiento Proteico-Postraduccional , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismoRESUMEN
A novel protein tyrosine kinase (PTK) was identified in Schistosoma mansoni and designated SmFes. SmFes exhibits the characteristic features of Fes/Fps/Fer (fes, feline sarcoma; fps, Fujinami poultry sarcoma; fer, fes related) PTKs, containing three coiled-coil regions, an SH2 (Src-homology-2) and a TK (tyrosine kinase catalytic) domain signature. SmFes is the first gene from the Fes/Fps/Fer family identified in S. mansoni, and is a single copy gene. Phylogenetic analyses revealed that SmFes is most closely related to its invertebrate orthologues. The assembly of the SmFes cDNA and genomic sequences indicated the presence of 18 introns in SmFes. Comparison of its genomic structure with those of human Fps/Fes and Drosophila Fps indicates that intron positions are conserved within the region encoding the kinase domain. Analysis of partial cDNA clones showed the presence of a 9 bp insertion at the 3' end of exon 10, producing two different cDNA populations, pointed as an alternative splicing event. In addition, an allele of SmFes containing a 15 bp insertion was observed in the genomic sequence. Quantitative RT-PCR indicated that the overall transcription level of SmFes is rather low in all parasite developmental stages. Moreover, SmFes mRNA levels decrease progressively after cercarial transformation, consistent with a role for the corresponding protein in the early stages of infection.
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Evolución Molecular , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/metabolismo , Schistosoma mansoni/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Protein Tyrosine Kinases (PTKs) are important molecules in intra- and inter-cellular communication, playing a major role in signal transduction processes. We have previously identified and characterized the molecular structure of a new PTK in Schistosoma mansoni, SmFes. SmFes exhibits the characteristic features of Fes/Fps protein tyrosine kinase subfamily of which it is the first member described in helminths. Herein, we show that genes orthologous to SmFes are also present in other Schistosoma species and the transcript is detected in Schistosoma japonicum. The SmFes protein was detected at all the main life-cycle stages and was most abundant in cercariae and newly-transformed schistosomula. However, no protein was detected in schistosomula maintained in vitro for 7 days. By immunolocalization assays we showed that SmFes is particularly concentrated at the terebratorium of miracidia and tegument of cercaria and schistosomula skin-stage. These findings suggest that SmFes may play a role in signal transduction pathways involved in larval transformation after penetration into intermediate and definitive hosts.
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Proteínas Proto-Oncogénicas c-fes/fisiología , Schistosoma mansoni/enzimología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Biomphalaria , Western Blotting , Secuencia Conservada , Femenino , Regulación Enzimológica de la Expresión Génica , Interacciones Huésped-Parásitos/fisiología , Masculino , Ratones , Modelos Estructurales , Proteínas Proto-Oncogénicas c-fes/biosíntesis , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/genética , Schistosoma mansoni/genética , Schistosoma mansoni/fisiologíaRESUMEN
Schistosoma mansoni signal transduction pathways are promising sources of target molecules for the development of novel control strategies against this platyhelminth parasite of humans. Members of the protein kinase C (PKC) family play key roles in such pathways activated by both receptor tyrosine kinases and other receptors, controlling a variety of physiological processes. Here, we report the cloning and molecular characterization of the first PKC identified in S. mansoni. Structural analysis indicated that SmPKC1 exhibits all the features typical of the conventional PKC subfamily. The gene structure was determined in silico and found to comprise a total of 15 exons and 14 introns. This structure is highly conserved; all intron positions are also present in the human PKCbeta gene and most of the exon sizes are identical. Using PCR on genomic DNA we were able to show that putative orthologues of SmPKC1 are present in 9 Schistosoma species. SmPKC1 expression is developmentally regulated with the highest level of transcripts in miracidia, whereas SmPKC1 protein expression is higher in the sporocyst. The localization of SmPKC1 on the sporocyst ridge cyton and in schistosomula acetabular glands suggests that the enzyme plays a role in signal transduction pathways associated with larval transformation.