RESUMO
This paper is the first report on the in vitro effects of licochalcone A, a chalcone isolated from Glycyrrhiza inflate Batalin (Leguminosae), on Schistosoma mansoni adult worms. In vitro, licochalcone A afforded lethal concentrations for 50% of parasites (LC50) of 9.12±1.1 and 9.52±0.9µM against female and male adult worms, respectively, at 24h. Additionally, the compound reduced the total number of S. mansoni eggs and affected the development of eggs produced by S. mansoni adult worms. Together, the results achieved after 24h showed that licochalcone A was 55.7- and 53.3-fold more toxic to S. mansoni female and male adult worms than to Chinese hamster ovary fibroblasts cells, respectively. Treatment with licochalcone A elicited drastic changes in the tegument of S. mansoni adult worms, as well as mitochondrial alteration and chromatin condensation. Licochalcone A also increased the superoxide anion level and decreased the superoxide dismutase activity in S. mansoni adult worms. Overall, our results indicated that licochalcone A displays in vitro schistosomicidal activity. This effect may result from increased production of reactive oxygen species (ROS) induced by the action of licochalcone A. The resulting ROS could act on the S. mansoni tegument and membranes and help induce the death of S. mansoni adult worms.
Assuntos
Chalconas/farmacologia , Glycyrrhiza , Extratos Vegetais/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Fatores Etários , Animais , Relação Dose-Resposta a Droga , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Óvulo/patologia , Extratos Vegetais/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Schistosoma mansoni/citologia , CaramujosRESUMO
BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.
Assuntos
Caseína Quinase II/metabolismo , DNA de Protozoário/metabolismo , Proteína HMGB1/metabolismo , Schistosoma mansoni/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citosol/metabolismo , DNA Super-Helicoidal/metabolismo , Ensaios Enzimáticos , Feminino , Granuloma/metabolismo , Proteína HMGB1/química , Proteína HMGB1/genética , Células HeLa , Humanos , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Mapas de Interação de Proteínas , Schistosoma mansoni/citologia , Schistosoma mansoni/ultraestruturaRESUMO
INTRODUCTION: Pirajá da Silva made a seminal contribution to helminthology by demonstrating both schistosome eggs in feces of patients from the State of Bahia and the morphology of Schistosoma mansoni adult worms. METHODS: In this study, a microscopic investigation of the whole-mounts deposited at the Helminthological Collection of the Oswaldo Cruz Institute is presented. Confocal laser scanning microscopy was used as the main investigation technique. RESULTS: At the anterior end of the adult male, suckers with well-developed musculature and germinative cells inside the testicular lobes were observed, as well as spines located in the mid region of the male gynecophoric canal. Tegumental tubercles and transversal and longitudinal muscular bundles were observed at the dorsal surface. The female reproductive system presented a uterus lacking eggs and an elongated ovary with germinative cells. The vitellaria were restricted to the extreme posterior end of each female connected to a short vitelline duct. CONCLUSIONS: The results reported in this study demonstrate that the characteristic studied here are similar to those previously reported, using fresh worms. Moreover, this study also highlights the importance of deposits of specimens in helminthological collections, which further permit revisiting whole-mounts in such institutions.
INTRODUÇÃO: Pirajá da Silva fez contribuição magnífica à helmintologia ao descrever ovos de Schistosoma mansoni nas fezes de um paciente, no Estado da Bahia e a morfologia de vermes adultos. MÉTODOS: Neste estudo, apresentamos uma avaliação microscópica das lâminas montadas e depositadas na Coleção Helmintológica do Instituto Oswaldo Cruz. A técnica empregada nesta nova análise foi a microscopia de varredura a laser confocal. RESULTADOS: Na parte anterior dos vermes adultos machos, observamos ventosas com musculatura bem desenvolvida e células germinativas dentro dos lobos testiculares. Visualizamos, também, espinhos localizados na região mediana do canal ginecóforo. Na superfície dorsal, encontramos tubérculos e feixes musculares transversais e longitudinais. Em relação ao aparelho reprodutivo feminino, pudemos distinguir um ovo no interior do útero e o ovário alongado com células germinativas. As glândulas vitelínicas estavam restritas à parte posterior das fêmeas conectadas por um ducto vitelínico curto. CONCLUSÕES: As características morfológicas são similares as estudadas anteriormente por Pirajá da Silva com vermes frescos. Além disso, este estudo demonstra a importância de se depositar espécimes nas coleções helmintológicas abrindo possibilidade de novos estudos com estas lâminas.
Assuntos
Animais , Feminino , Masculino , Schistosoma mansoni/citologia , Microscopia ConfocalRESUMO
BACKGROUND: Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The Schistosoma mansoni genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the S. mansoni predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets. RESULTS: We have identified 252 ePKs, which corresponds to 1.9% of the S. mansoni predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that S. mansoni has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in S. mansoni or belong to an expanded family in this parasite. Only 16 S. mansoni ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite. CONCLUSIONS: Our approach has improved the functional annotation of 40% of S. mansoni ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of S. mansoni in response to diverse environments during the parasite development, vector interaction, and host infection.
Assuntos
Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Proteômica , Schistosoma mansoni/enzimologia , Schistosoma mansoni/parasitologia , Animais , Domínio Catalítico , Cadeias de Markov , Filogenia , Proteínas Quinases/química , Proteoma/química , Proteoma/classificação , Proteoma/metabolismo , Schistosoma mansoni/citologia , Transdução de SinaisRESUMO
INTRODUCTION: Pirajá da Silva made a seminal contribution to helminthology by demonstrating both schistosome eggs in feces of patients from the State of Bahia and the morphology of Schistosoma mansoni adult worms. METHODS: In this study, a microscopic investigation of the whole-mounts deposited at the Helminthological Collection of the Oswaldo Cruz Institute is presented. Confocal laser scanning microscopy was used as the main investigation technique. RESULTS: At the anterior end of the adult male, suckers with well-developed musculature and germinative cells inside the testicular lobes were observed, as well as spines located in the mid region of the male gynecophoric canal. Tegumental tubercles and transversal and longitudinal muscular bundles were observed at the dorsal surface. The female reproductive system presented a uterus lacking eggs and an elongated ovary with germinative cells. The vitellaria were restricted to the extreme posterior end of each female connected to a short vitelline duct. CONCLUSIONS: The results reported in this study demonstrate that the characteristic studied here are similar to those previously reported, using fresh worms. Moreover, this study also highlights the importance of deposits of specimens in helminthological collections, which further permit revisiting whole-mounts in such institutions.
Assuntos
Schistosoma mansoni/citologia , Animais , Feminino , Masculino , Microscopia ConfocalRESUMO
The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separeted sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59 percent) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33 percent females had an incipient ovary (without cellular differentiation). At week four, 77.2 percent males presented testicular lobes with few germinative cells while 26 percent had developing tegumental tubercles. The immature ovary was observed in 69 percent females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.
Assuntos
Animais , Feminino , Masculino , Camundongos , Gônadas/crescimento & desenvolvimento , Maturidade Sexual , Schistosoma mansoni/crescimento & desenvolvimento , Microscopia Confocal , Schistosoma mansoni/citologia , Fatores de TempoRESUMO
Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85 percent. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4 percent and Neutral Red 1 percent). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.
Assuntos
Animais , Camundongos , Corantes , Corantes Fluorescentes , Óvulo/crescimento & desenvolvimento , Schistosoma mansoni/citologia , Schistosoma mansoni/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Laranja de Acridina , Óvulo/citologia , Azul TripanoRESUMO
The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separated sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.
Assuntos
Gônadas/crescimento & desenvolvimento , Schistosoma mansoni/crescimento & desenvolvimento , Maturidade Sexual , Animais , Feminino , Masculino , Camundongos , Microscopia Confocal , Schistosoma mansoni/citologia , Fatores de TempoRESUMO
Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.
Assuntos
Corantes , Corantes Fluorescentes , Óvulo/crescimento & desenvolvimento , Schistosoma mansoni/citologia , Schistosoma mansoni/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Laranja de Acridina , Animais , Camundongos , Óvulo/citologia , Azul TripanoRESUMO
Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. The presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation.
Assuntos
Apirase/análise , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Apirase/genética , Apirase/imunologia , Clonagem Molecular , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Schistosoma mansoni/citologia , Homologia de Sequência de AminoácidosRESUMO
A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.
Assuntos
Proteínas de Bactérias , DNA de Helmintos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Helminto/metabolismo , RNA de Helmintos/metabolismo , Schistosoma mansoni/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Helminto/química , Sondas de Oligonucleotídeos , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/citologia , Schistosoma mansoni/genética , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
The authors present morphogenetic and biomechanical approaches on the concept of the Schistosoma mansoni granulomas, considering them as organoid structures that depend on cellular adhesion and sorting, forming rearrangement into hierarchical concentric layers, creating tension-dependent structures, aiming to acquire round form, since this is the minimal energy form, in which opposing forces pull in equally from all directions and are in balance. From the morphogenetic point of view, the granulomas function as little organs, presenting maturative and involutional stages in their development with final disappearance (pre-granulomatous stages, subdivided in: weaky and/or initial reactive and exudative; granulomatous stages: exudative-productive, productive and involutional). A model for the development of granulomas was suggested according to the following stages: encapsulating, focal hystolysis, fiber production, orientation and compacting and involution and desintegration. The autors concluded that schistosomal granuloma is not a tangled web of individual cells and fibers, but an organized structure composed by host and parasite components, which is not formed to attack the miracidia, but functions as an hybrid interface between two different phylogenetic beings.
Assuntos
Animais , Fenômenos Biomecânicos , Matriz Extracelular/parasitologia , Fibrose , Granuloma , Morfogênese/fisiologia , Schistosoma mansoni/citologia , Microscopia ConfocalRESUMO
A computer software for image analysis (IMAGE PRO PLUS, MEDIA CYBERNETICS) was utilized in male and females adult worms, aiming the morphological characterization of Schistosoma mansoni samples isolated from a slyvatic rodent, Nectomys squamipes, and humans in Sumidouro, Rio de Janeiro, Brazil and recovered from Mus musculus C3H/He. The following characters for males's testicular lobes were analyzed: number, area, density, larger and smaller diameter, longer and shorter axis and perimeter and extension; for females: area, longer and shorter axis, larger and smaller diameter and perimeter of the eggs and spine; oral and ventral suckers area and distance between them in both sex were determined. By the analysis of variance (one way ANOVA) significant differences (p < 0.05) were observed in all studied characters, except for the density of testicular lobes. Significant differences (p < 0.05) were detected for all characters in the female worms. Data ratify that sympatric isolates present phenotypic differences and the adult female characters are useful for the proper identification of S. mansoni isolates.
Assuntos
Schistosoma mansoni/citologia , Análise de Variância , Animais , Feminino , Masculino , Camundongos , Roedores , Schistosoma mansoni/isolamento & purificaçãoRESUMO
Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss). The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.
Assuntos
Sistema Digestório/citologia , Microscopia Confocal , Schistosoma mansoni/citologia , Sistema Urogenital/citologia , Animais , Humanos , Masculino , CamundongosRESUMO
Estudou-se os níveis de anticorpo letal contra esquistossômulos "in vitro" em pacientes infectados com S. mansoni, na tentativa de estabelecer seu significado biológico.[...]