RESUMO
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
We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.
Assuntos
Doença de Chagas/complicações , Coxiella burnetii , Febre Q/complicações , Trypanosoma cruzi , Células Vero/microbiologia , Animais , Doença de Chagas/transmissão , Chlorocebus aethiops , Progressão da Doença , Concentração de Íons de Hidrogênio , Microscopia Confocal , Vacúolos/metabolismo , Células Vero/parasitologia , Células Vero/ultraestruturaRESUMO
OBJECTIVES: This study was performed to investigate the activity of tamoxifen, an antioestrogen widely used in the treatment of breast cancer, against Leishmania. METHODS: Drug activity was assessed in vitro against axenically grown promastigotes and amastigotes through cell counting or by measuring the cleavage of MTT, and against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of intracellular parasites. Intravacuolar pH changes induced inside parasitophorous vacuoles of Leishmania (Leishmania) amazonensis-infected macrophages were evaluated using the fluorescent probes SNAFL-calcein and Acridine Orange. RESULTS: Tamoxifen killed L. (L.) amazonensis promastigotes and amastigotes with 50% inhibitory concentrations (IC50) of 16.4 +/- 0.2 and 11.1 +/- 0.2 microM, respectively. The drug was also effective against Leishmania (Viannia) braziliensis, Leishmania (Leishmania) major, Leishmania (Leishmania) chagasi and Leishmania (Leishmania) donovani with IC(50) values ranging from 9.0 to 20.2 microM. Tamoxifen induced a rapid and long-lasting alkalinization of the vacuolar environment. We also provide evidence that tamoxifen is more effective against promastigotes and amastigotes at pH 7.5 when compared with cultures at pH 4.5. CONCLUSIONS: Tamoxifen effectively kills several Leishmania species and its activity against the parasite is increased by a modulation of the host cell intravacuolar pH induced by the drug.
Assuntos
Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Antiprotozoários , Leishmania mexicana/efeitos dos fármacos , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Vacúolos/química , Álcalis , Animais , Sobrevivência Celular/efeitos dos fármacos , Estradiol/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Vacúolos/efeitos dos fármacos , Vacúolos/parasitologiaRESUMO
Calomys callosus (Rodentia: Cricetidae) chronically infected with CL strain of Trypanosoma cruzi undergo recrudescence of the acute phase when treated with the immunosuppressor cyclophosphamide. The distribution of cytoskeletal proteins in cardiac tissue of immunosuppressed animals was mapped by immunofluorescence and electron microscopy to evaluate myofibrillar distribution during the intracellular life cycle of T. cruzi. Cardiac muscle sections showed enhancement of myocarditis and parasite proliferation after immunosuppression. Immunofluorescence using monoclonal antibodies against myosin, actin, desmin, titin, tropomyosin, and troponin T demonstrated disruption and loss of contractile proteins, such as myosin and actin. Desmin and titin were irregularly distributed in close proximity to parasite nests. Ultrastructural observations confirmed alterations of cardiac cells with Z-line fragmentation, indistinguishable I-bands and A-bands, and loss of myofibrillar elements. The disruption of the muscle cell architecture was greater as infection progressed, probably as a result of increased myocarditis and physical displacement due to the activity of flagellated parasites.
Assuntos
Arvicolinae/parasitologia , Cardiomiopatia Chagásica/tratamento farmacológico , Ciclofosfamida/uso terapêutico , Modelos Animais de Doenças , Imunossupressores/uso terapêutico , Miocárdio/patologia , Trypanosoma cruzi/patogenicidade , Animais , Animais não Endogâmicos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Doença Crônica , Feminino , Humanos , Microscopia Confocal , Microscopia Eletrônica , Músculo Esquelético/química , Músculo Esquelético/patologia , Miocárdio/química , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/parasitologia , Doenças dos Roedores/patologiaRESUMO
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
Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.