RESUMO
It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.
Assuntos
Humanos , Membrana Celular/parasitologia , Citoesqueleto/parasitologia , Trypanosoma cruzi/fisiologia , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Células HeLa/parasitologia , Células HeLa/ultraestrutura , Fatores de TempoRESUMO
It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.
Assuntos
Membrana Celular/parasitologia , Citoesqueleto/parasitologia , Trypanosoma cruzi/fisiologia , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Células HeLa/parasitologia , Células HeLa/ultraestrutura , Humanos , Fatores de TempoRESUMO
The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii suggests that these parasites might explore similar strategies to complete their life cycles. T. cruzi is the etiological agent of the life-threatening Chagas' disease, whereas T. dionisii is a bat trypanosome and probably not capable of infecting humans. Here we sought to compare mammalian cell invasion and intracellular traffic of both trypanosomes and determine the differences and similarities in this process. The results presented demonstrate that T. dionisii is highly infective in vitro, particularly when the infection process occurs without serum and that the invasion is similarly affected by agents known to interfere with T. cruzi invasion process. Our results indicate that the formation of lysosomal-enriched compartments is part of a cell-invasion mechanism retained by related trypanosomatids, and that residence and further escape from a lysosomal compartment may be a common requisite for successful infection. During intracellular growth, parasites share a few epitopes with T. cruzi amastigotes and trypomastigotes. Unexpectedly, in heavily infected cells, amastigotes and trypomastigotes were found inside the host cell nucleus. These findings suggest that T. dionisii, although sharing some features in host cell invasion with T. cruzi, has unique behaviors that deserve to be further explored.
Assuntos
Núcleo Celular/parasitologia , Trypanosoma/crescimento & desenvolvimento , Animais , Linhagem Celular , Lisossomos/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Vacúolos/parasitologiaRESUMO
Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a highly prevalent vector-borne disease in Latin America. Chagas' disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host's blood, infected triatomines release in their feces highly motile and infective metacyclic trypomastigotes that may initiate infection. Metacyclic trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellularly, into amastigote-like forms. Unlike their intracellular counterparts, these amastigote-like forms, henceforth called amastigotes, are capable of infecting host cells. Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.
Assuntos
Actinas/metabolismo , Estágios do Ciclo de Vida/fisiologia , Trypanosoma cruzi/fisiologia , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Chlorocebus aethiops , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Trypanosoma cruzi/ultraestrutura , Células VeroRESUMO
Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae-defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana/fisiologia , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/fisiologia , Actinas/metabolismo , Adesinas Bacterianas/química , Sequência de Aminoácidos , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fenótipo , Fosforilação , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.
Assuntos
Colesterol/metabolismo , Trypanosoma cruzi/patogenicidade , Células Vero/parasitologia , Animais , Chlorocebus aethiops , Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/metabolismo , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Trypanosoma cruzi/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.
Assuntos
Núcleo Celular , Citoplasma/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Linhagem Celular , Feminino , Fibroblastos/parasitologia , Estágios do Ciclo de Vida , Camundongos , Microscopia de FluorescênciaRESUMO
Previous studies have shown that Coxiella burnetii, an intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics, is an effective modulator of the intracellular traffic of trypomastigote forms of Trypanosoma cruzi. In addition, vacuolar and cellular pH are related to fusion events that result in doubly infected phagosomes. T. cruzi, the etiological agent of Chagas' disease, occurs as different strains grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle, and T. cruzi II, linked to the human disease. In this work we compared extracellular amastigotes (EA), metacyclic trypomastigotes (MT) and tissue culture derived trypomastigotes (TCT) belonging to T. cruzi I or T. cruzi II for their ability to invade and escape from their parasitophorous vacuole (PV), in Vero cells or Vero cells harboring the bacterium, C. burnetti. Distinct invasion patterns were observed between different infective stages and between infective forms of different strains. Studies on the transference kinetics revealed that pH modulates the intracellular traffic of each infective stage, but this influence is not exclusive for each phylogenetic group. Endosomal to lysosomal sequential labeling with EEA-1 and LAMP-1 of the PV formed during the entry of each infective form revealed that the phagosome maturation processes are distinct but not strain-dependent. Due to their low hemolysin and trans-sialidase activities, MTs are retained for longer periods in LAMP-1 positive vacuoles. Our results thus suggest that despite the contrasting invasion capabilities, parasites of distinct phylogenetic group behave in similar fashion once inside the host cell.
Assuntos
Coxiella burnetii/fisiologia , Trypanosoma cruzi/fisiologia , Vacúolos/parasitologia , Animais , Chlorocebus aethiops , Endossomos/química , Endossomos/parasitologia , Concentração de Íons de Hidrogênio , Proteína 1 de Membrana Associada ao Lisossomo/análise , Lisossomos/química , Lisossomos/parasitologia , Proteínas de Membrana/análise , Microscopia de Fluorescência , Células Vero , Proteínas de Transporte Vesicular/análiseRESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.
Assuntos
Citoplasma/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Células Cultivadas/parasitologia , Chlorocebus aethiops , Citoplasma/ultraestrutura , Células HeLa/parasitologia , Humanos , Microscopia Eletrônica de Transmissão , Filogenia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Células Vero/parasitologiaRESUMO
O agente etiológico da doença de Chagas, Trypanosoma cruzi, ocorre como cepas ou isolados que podem ser agrupados em duas grandes linhagens filogenéticas: T. cruzi I associada ao ciclo silvestre e T. cruzi II ligada à doença humana. No hospedeiro mamífero o parasita tem que invadir células, e vários estudos relacionam as formas flageladas tripomastigotas neste processo. Diferentes componentes de superfície dos parasitas e alguns dos respectivos receptores foram identificados. Em nosso trabalho temos procurado compreender como amastigotas, que normalmente são encontrados crescendo no citoplasma, podem invadir células de mamíferos com infectividade comparável às dos tripomastigotas. Encontramos diferenças nas respostas celulares induzidas por amastigotas e tripomastigotas em relação a componentes de citoesqueleto e projeções de membrana ricas em actina. Amastigotas de cepas de T. cruzi I gerados extracelularmente, podem apresentar infectividade maior que tripomastigotas metacíclicos para linhagens celulares e células com expressão alterada em diferentes classes de componentes celulares. Células albergando a bactéria Coxiella burnetii tem nos permitido obter novos enfoques sobre as propriedades de tráfego intracelular das diferentes formas infectivas do T. cruzi, revelando requerimentos inesperados para o parasita transitar entre seu vacúolo parasitóforo até seu destino final no citoplasma da célula hospedeira.
Assuntos
Humanos , Animais , Citoplasma , Trypanosoma cruzi , Células Cultivadas , Chlorocebus aethiops , Células HeLa , Microscopia Eletrônica , Filogenia , Células VeroRESUMO
Analisar as manifestaçöes cardiocirculatórias do hipertireoidismo em idosos. Vinte e quatro portadores de hipertireoidismo com idades variando de 60 a 87 (média 73,5) anos, 18 do sexo feminino. A análise cardiocirculatória foi realizada através dos exames clínico, eletrocardiográfico, radiológico, fonomecanocardiográfico e ecocardiográfico. Manifestaçöes cardiocirculatórias foram observadas em 17 (70,9%) idosos, nove (37,5%) com insuficiência cardíaca. Alteraçöes eletrocardiográficas foram evidenciadas em 20 (83,8%) casos, predominando as taquiarritmias (62,5%) fibrilaçäo atrial crônica em oito (33,3%) e taquicardia sinusal em cinco (20,8%). Analisando-se separadamente os pacientes com e sem doença cardiocirculatória associada, näo se observaram diferenças estatisticamente significativas nos vários parâmetros eletrocardiográficos estudados. A freqüêmcia de cardiomegalia foi estatisticamente mais elevada nos hipertireoideos, tanto portadores (64,7%) como näo portadores (57,1%) de afecçöes cardiocirculatórias associadas, em relaçäo aos idosos normais (23,9%). O desempenho ventricular esquerdo avaliado em 14 pacientes, através do quociente sistólico mostrou-se normal ou elevado em 12 (85,7%). O percentual de encurtamento da fibra (delta D%) foi maior que 30% em todos os casos em que foi determinado. Nenhum paciente apresentou ao ecocardiograma alteraçöes sugestivas de cardiomiopatia hipertrófica simétrica ou assimétrica e de prolapso mitra. O hipertireoidismo no idoso determina ...