RESUMO
Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies.
Assuntos
Estágios do Ciclo de Vida , Toxoplasma/crescimento & desenvolvimento , Gravação em Vídeo , Animais , Feminino , Humanos , Gravidez , Prevalência , Toxoplasmose/complicações , Toxoplasmose/parasitologiaRESUMO
BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.
Assuntos
Bacteroides fragilis/enzimologia , Bacteroides fragilis/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Fosfopiruvato Hidratase , Plasminogênio , Vesículas ExtracelularesRESUMO
BACKGROUND: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.
Assuntos
Bacteroides fragilis/enzimologia , Vesículas Extracelulares/enzimologia , Fosfopiruvato Hidratase/análise , Bacteroides fragilis/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Vesículas Extracelulares/ultraestrutura , Humanos , Laminina , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Fosfopiruvato Hidratase/metabolismo , PlasminogênioRESUMO
This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in embryonated eggs, larval stages and in the adults' survival. As most current anthelmintics are not effective against all nematode life stages, this protocol can contribute to the identification of new alternatives to helminthic infections (Sant' Anna et al., 2016 ).
RESUMO
Dinitroanilines are known herbicides that impair the polymerization of microtubules. This study investigated the effects of oryzalin and trifluralin on the viability, morphology, and ultrastructure of different life stages of Caenorhabditis elegans. Both drugs reduced the survival of the adult population in 50% after three days of treatment with concentrations of approximately 30 µM and 57 µM, respectively. The development of new adults was monitored for seven days and treatment with both drugs also showed a decrease in the adult population. 25 µM Oryzalin or trifluralin inhibited the hatching of eggs by nearly 100%. Both drugs showed remarkable larvicidal activity at 25 µM against the larvae at first and second stages (L1-L2) and at third and fourth stages (L3-L4) after 24 h. Treatment with dinitroanilines led to incomplete egg embryo development. The oryzalin and trifluralin treatments caused the detachment of cuticular layers of adults and larvae and the formation of a large number of intracellular membrane whirls and lipid bodies in the hypodermal cells and non-contractile muscles of adults. Both drugs also provoked the bagging process, which generated lesions in the uterus of the adults. In addition, trifluralin caused the detachment of certain areas of the cuticle adjacent to the hypodermis in a large number of nematodes. Our results suggest that dinitroanilines are a potentially new alternative for anthelmintic chemotherapy.
Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Dinitrobenzenos/farmacologia , Herbicidas/farmacologia , Sulfanilamidas/farmacologia , Trifluralina/farmacologia , Moduladores de Tubulina/farmacologia , Albendazol/farmacologia , Análise de Variância , Animais , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/ultraestrutura , Larva/efeitos dos fármacos , Larva/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Óvulo/efeitos dos fármacos , Óvulo/ultraestruturaRESUMO
Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite's DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13-25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition--with the appearance of 'tethered' parasites--malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis.
Assuntos
Ciprofloxacina/agonistas , Ésteres/administração & dosagem , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/tratamento farmacológico , Animais , Antimaláricos/administração & dosagem , Antimaláricos/farmacologia , Divisão Celular/efeitos dos fármacos , Ciprofloxacina/administração & dosagem , Ciprofloxacina/farmacologia , Ésteres/farmacologia , Feminino , Camundongos , Análise de Sobrevida , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologiaRESUMO
Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Bacteroides fragilis/fisiologia , Adesinas Bacterianas/genética , Animais , Bacteroides fragilis/patogenicidade , Western Blotting , Técnicas de Inativação de Genes , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Viabilidade Microbiana , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
This study investigated the effects of albendazole on the viability, morphology and ultrastructure of different life stages of Caenorhabditis elegans. The albendazole EC50 value after seven days of treatment was 18.43 µM. This concentration was very efficient against all the stages. Light and electron microscopy analysis showed damage to the body wall of the adults and larvae. An intense desquamation of the cuticle of larvae and of the surface of the eggs was observed, preventing their hatching and development. The main ultrastructural damage detected was the degeneration of the mitochondria in the noncontractile muscle of the body wall, which appeared as large vacuoles. This study reaffirmed the use of C. elegans as a screening system for compounds with potential anthelmintic activity and showed the effects of albendazole on the different life stages of these worms.
Assuntos
Albendazol/farmacologia , Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Análise de Variância , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Larva/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Toxoplasma gondii is an important opportunistic pathogen for immunocompromised patients and responsible for toxoplasmic encephalitis, which is often lethal. Treatment for this infection is limited to a restricted therapeutic arsenal. In this work we tested the combination of fluconazole with the current treatment for acute toxoplasmosis on the murine model in vivo. Different experimental groups were treated with combinations of sulfadiazine plus pyrimethamine with fluconazole and pyrimethamine with fluconazole. Fluconazole is an important antifungal triazole used against others CNS related opportunistic pathogens such as Cryptococcus neoformans and Candida spp. The combinations of fluconazole plus sulfadiazine and pyrimethamine or fluconazole plus pyrimethamine were remarkably effective against T. gondii in vivo. The 10-day treatment with 10mg/kg/day of fluconazole combined with 40/1mg/kg/day sulfadiazine and pyrimethamine resulted in 93% survival of CF1 mice acutely infected with the highly virulent T. gondii RH strain, versus 36% of mice treated with just sulfadiazine and pyrimethamine. Combinations of fluconazole with lower doses of sulfadiazine and pyrimethamine or with just pyrimethamine were also efficient in reducing the mortality of mice compared with the treatment without fluconazole. The results obtained are promising for the treatment of human toxoplasmosis and point to the need to extend these studies to other murine models.
Assuntos
Antiprotozoários/uso terapêutico , Fluconazol/uso terapêutico , Pirimetamina/uso terapêutico , Sulfadiazina/uso terapêutico , Toxoplasmose Animal/tratamento farmacológico , Doença Aguda , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Antiprotozoários/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Fluconazol/farmacologia , Concentração Inibidora 50 , Macaca mulatta , Camundongos , Pirimetamina/farmacologia , Sulfadiazina/farmacologia , Toxoplasma/efeitos dos fármacosRESUMO
The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.