RESUMO
Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.
Assuntos
Giardia/isolamento & purificação , Giardíase/parasitologia , Isópteros/parasitologia , Animais , Brasil , Fezes/parasitologia , Flagelos/ultraestrutura , Giardia/classificação , Giardia/ultraestrutura , Giardíase/transmissão , Microscopia Eletrônica de Varredura , Organelas/ultraestrutura , Trofozoítos/citologiaRESUMO
Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.
Assuntos
Antiprotozoários/farmacologia , Giardia lamblia/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Trofozoítos/efeitos dos fármacos , Células CACO-2 , Citocinese/efeitos dos fármacos , Giardia lamblia/citologia , Giardia lamblia/crescimento & desenvolvimento , Humanos , Concentração Inibidora 50 , Microscopia , Microscopia Eletrônica , Testes de Sensibilidade Parasitária , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimentoRESUMO
Based on morphological, morphometric, and molecular data, we describe a new hemoparasite of the genus Haemogregarina Danilewsky 1885, isolated from the Brazilian aquatic turtle Podocnemis unifilis (Testudines: Podocnemididae). The new species, Haemogregarina podocnemis sp. nov. (Apicomplexa: Haemogregarinidae), is characterized by small trophozoites with a single cytoplasmic vacuole on one side; pre-meronts with nuclear chromatin dispersed in the cytoplasm, with or without cytoplasmic vacuoles; meronts that are usually broad and slightly curved (kidney-shaped), with an average of eight small rectangular nuclei; immature gamonts (bean-shaped) with two morphological types: one with nuclear chromatin dispersed in the cytoplasm and the other with nuclei in the middle of the cell; mature gamonts of two morphological types: one with a length equal to or greater than that of the erythrocyte and the width of the nuclei similar to that of the hemoparasite and the other smaller than the erythrocyte with the width of the nuclei less than that of the hemoparasite. This is the first hemogregarine species described that infects the Brazilian turtle Po. unifilis. These findings highlight the need for further studies of Haemogregarina spp. to better determine the biodiversity of this understudied parasite group.
Assuntos
Coccidiose/veterinária , Eucoccidiida/classificação , Eucoccidiida/isolamento & purificação , Trofozoítos/ultraestrutura , Animais , Brasil , Coccidiose/parasitologia , Eritrócitos/parasitologia , Eucoccidiida/genética , Feminino , Masculino , Microscopia , RNA Ribossômico 18S/genética , Rios , Trofozoítos/citologia , Tartarugas/parasitologiaRESUMO
The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.
Assuntos
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Genes de Protozoários/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Morte Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes Reporter , Humanos , Fagocitose , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Trofozoítos/citologia , Trofozoítos/metabolismo , Fatores de Virulência/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Giardia lamblia is a worldwide protozoan responsible for a significant number of intestinal infections. There are several drugs for the treatment of giardiasis, but they often cause side effects. Curcumin, a component of turmeric, has antigiardial activity; however, the molecular target and mechanism of antiproliferative activity are not clear. The effects of curcumin on cellular microtubules have been widely investigated. Since tubulin is the most abundant protein in the cytoskeleton of Giardia, to elucidate whether curcumin has activity against the microtubules of this parasite, we treated trophozoites with curcumin and the cells were analyzed by scanning electron microscopy and confocal microscopy. Curcumin inhibited Giardia proliferation and adhesion in a time-concentration-dependent mode. The higher inhibitory concentrations of curcumin (3 and 15µM) disrupted the cytoskeletal structures of trophozoites; the damage was evident on the ventral disk, flagella and in the caudal region, also the membrane was affected. The immunofluorescence images showed altered distribution of tubulin staining on ventral disk and flagella. Additionally, we found that curcumin caused a clear reduction of tubulin expression. By docking analysis and molecular dynamics we showed that curcumin has a high probability to bind at the interface of the tubulin dimer close to the vinblastine binding site. All the data presented indicate that curcumin may inhibit Giardia proliferation by perturbing microtubules.
Assuntos
Curcumina/farmacologia , Giardia lamblia/efeitos dos fármacos , Trofozoítos/efeitos dos fármacos , Animais , Flagelos , Microscopia Eletrônica de Varredura , Microtúbulos/fisiologia , Trofozoítos/citologia , Tubulina (Proteína)/metabolismoRESUMO
The chemotherapeutic agents used for the treatment of giardiasis are often associated with adverse side effects and are refractory cases, due to the development of resistant parasites. Therefore the search for new drugs is required. We have previously reported the giardicidal effects of metronidazole (MTZ) and its analogues (MTZ-Ms, MTZ-Br, MTZ-N(3), and MTZ-I) on the trophozoites of Giardia lamblia. Now we evaluated the activity of some giardicidal MTZ analogues in experimental infections in gerbils and its effects on the morphology and ultrastructural organization of Giardia. The giardicidal activity in experimental infections showed ED(50) values significantly lower for MTZ-I and MTZ-Br when compared to MTZ. Transmission electron microscopy was employed to approach the mechanism(s) of action of MTZ analogues upon the protozoan. MTZ analogues were more active than MTZ in changing significantly the morphology and ultrastructure of the parasite. The analogues affected parasite cell vesicle trafficking, autophagy, and triggered differentiation into cysts. These results coupled with the excellent giardicidal activity and lower toxicity demonstrate that these nitroimidazole derivates may be important therapeutic alternatives for combating giardiasis. In addition, our results suggest a therapeutic advantage in obtaining synthetic metronidazole analogues for screening of activities against other infectious agents.
Assuntos
Antiprotozoários/farmacologia , Giardia lamblia/efeitos dos fármacos , Giardíase/parasitologia , Metronidazol/análogos & derivados , Análise de Variância , Animais , Linhagem Celular , Gerbillinae , Giardia lamblia/citologia , Giardia lamblia/ultraestrutura , Concentração Inibidora 50 , Metronidazol/farmacologia , Microscopia Eletrônica de Transmissão , Carga Parasitária , Trofozoítos/citologia , Trofozoítos/efeitos dos fármacos , Trofozoítos/ultraestruturaRESUMO
Some structural observations on cultured Vahlkampfia sp. trophozoites are reported. Trophozoites are active and pleomorphic, producing large cell protrusions related to locomotion such as lamellipodia, filopodia and endocytic structures formed by hyaline cytoplasm, in which actin provides a framework that allows rapid changes in morphology. As observed by transmission electron microscopy, the cytoplasm is highly granular masking some cell organelles and the major cytoplasmic membrane systems. The structure of cell organelles such as the nucleus, endoplasmic reticulum, and digestive vacuoles is described. A common finding was the presence of 50 nm electron-dense round granules that are not limited by a membrane and that appear scattered in the cytoplasm, and whose function remains unknown. Apparently, the cell reserve material is glycogen, since complete trophozoites were positive to Schiff periodic-acid technique.
Assuntos
Amebíase/parasitologia , Ceratite/parasitologia , Schizopyrenida/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Polissacarídeos/análise , Schizopyrenida/citologia , Schizopyrenida/crescimento & desenvolvimento , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestruturaRESUMO
Entamoeba histolytica trophozoites can induce host cell apoptosis, which correlates with the virulence of the parasite. This phenomenon has been seen during the resolution of an inflammatory response and the survival of the parasites. Other studies have shown that E. histolytica trophozoites undergo programmed cell death (PCD) in vitro, but how this process occurs within the mammalian host cell remains unclear. Here, we studied the PCD of E. histolytica trophozoites as part of an in vivo event related to the inflammatory reaction and the host-parasite interaction. Morphological study of amoebic liver abscesses showed only a few E. histolytica trophozoites with peroxidase-positive nuclei identified by terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick end labelling (TUNEL). To better understand PCD following the interaction between amoebae and inflammatory cells, we designed a novel in vivo model using a dialysis bag containing E. histolytica trophozoites, which was surgically placed inside the peritoneal cavity of a hamster and left to interact with the host's exudate components. Amoebae collected from bags were then examined by TUNEL assay, fluorescence-activated cell sorting (FACS) and transmission electron microscopy. Nuclear condensation and DNA fragmentation of E. histolytica trophozoites were observed after exposure to peritoneal exudates, which were mainly composed of neutrophils and macrophages. Our results suggest that production of nitric oxide by inflammatory cells could be involved in PCD of trophozoites. In this modified in vivo system, PCD appears to play a prominent role in the host-parasite interaction and parasite cell death.
Assuntos
Apoptose , Cricetinae , Modelos Animais de Doenças , Entamoeba histolytica/citologia , Entamoeba histolytica/crescimento & desenvolvimento , Abscesso Hepático Amebiano/parasitologia , Animais , Fragmentação do DNA , Entamoeba histolytica/patogenicidade , Interações Hospedeiro-Parasita , Humanos , Abscesso Hepático Amebiano/imunologia , Macrófagos/imunologia , Masculino , Neutrófilos/imunologia , Óxido Nítrico/imunologia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , VirulênciaRESUMO
Iron is essential for nearly all organisms; in mammals, it is part of proteins such as haemoglobin, and it is captured by transferrin and lactoferrin. Transferrin is present in serum, and lactoferrin is secreted by the mucosa and by neutrophils at infection sites, as a host iron-withholding response, sequestering iron away from invading microorganisms. Additionally, all cells contain ferritin, which sequesters iron when its intracellular levels are increased, detoxifying and preventing damage. Liver ferritin contains 50% of iron corporal reserves. During evolution, pathogens have evolved diverse strategies to obtain iron from their hosts in order to survive. The protozoan Entamoeba histolytica invades the intestinal mucosa, causing dysentery, and the trophozoites often travel to the liver producing hepatic abscesses; thus, intestine and liver proteins could be important iron supplies for E. histolytica. We found that E. histolytica trophozoites can grow in both ferrous and ferric iron, and that they can use haemoglobin, holo-transferrin, holo-lactoferrin, and ferritin as in vitro iron sources. These proteins supported the amoeba growth throughout consecutive passages, similarly to ferric citrate. By confocal microscopy and immunoblotting, iron-binding proteins were observed specifically bound to the amoeba surface, and they were endocytosed, trafficked through the endosomal/lysosomal route, and degraded by neutral and acidic cysteine-proteases. Transferrin and ferritin were mainly internalized through clathrin-coated vesicles, and holo-lactoferrin was mainly internalized by caveola-like structures. In contrast, apo-lactoferrin bound to membrane lipids and cholesterol, inducing cell death. The results suggest that in vivo trophozoites secrete products that can destroy enterocytes, erythrocytes, and hepatocytes, releasing transferrin, haemoglobin, ferritin, and other iron-containing proteins, which, together with lactoferrin derived from neutrophils and acinar cells, could be used as abundant iron supplies by amoebas.
Assuntos
Endocitose , Entamoeba histolytica/fisiologia , Entamebíase/metabolismo , Entamebíase/parasitologia , Proteínas de Ligação ao Ferro/metabolismo , Trofozoítos/fisiologia , Animais , Bactérias/metabolismo , Bactérias/patogenicidade , Entamoeba histolytica/citologia , Entamoeba histolytica/patogenicidade , Entamebíase/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ferro/metabolismo , Microscopia Confocal , Trofozoítos/citologia , VirulênciaRESUMO
In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.
Assuntos
Citoesqueleto de Actina/metabolismo , Fibronectinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Trofozoítos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Depsipeptídeos/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/metabolismo , Entamoeba histolytica/ultraestrutura , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Tiazolidinas/farmacologia , Trofozoítos/citologia , Trofozoítos/efeitos dos fármacosRESUMO
Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Entamoeba histolytica/ultraestrutura , Miosinas/análise , Citoesqueleto de Actina/química , Animais , Citocinese , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Entamoeba histolytica/química , Entamoeba histolytica/citologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Trofozoítos/química , Trofozoítos/citologia , Trofozoítos/ultraestruturaRESUMO
The effects of sterol biosynthesis inhibitors on growth and fine structure of Giardia lamblia P1 strain cultures were analyzed. Azasterols demonstrated high efficacy in killing cells. The IC(50) values for 22,26-azasterol and 24(R,S),25-epiminolanosterol were 7muM and 170nM, respectively. Morphological analysis showed that azasterols induced changes in G. lamblia ultrastructure. The most significant alterations were: (a) considerable increase of the size of the peripheral vesicles, which are part of the parasite endosomal-lysosomal system; (b) appearance of autophagosomal structures; and (c) induction of differentiation, followed by an abnormal enlargement of encystation secretory vesicles. We propose that azasterols are effective chemotherapeutic drugs against Giardia lamblia in vitro and may have another target in cells besides sterol biosynthesis.