Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/22767
Title: Trypanosoma cruzi intracellular amastigotes isolated by nitrogen decompression are capable of endocytosis and cargo storage in reservosomes
Authors: Batista, Cassiano Martin
Kessler, Rafael Luis
Eger, Iriane
Soares, Maurilio José
Affilliation: Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / Universidade Estadual de Ponta Grossa. Departamento de Biologia Geral. Ponta Grossa, Paraná, Brazil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.
Abstract: Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10(7) amastigotes--with typical shape and positive for the amastigote marker Ssp4--from 5x10(6) infected Vero cells (48 h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.
Keywords: Chagas Disease
Intracellular Space
Endocytosis
Mammals
Keywords in spanish: Enfermedad de Chagas
Espacio Intracelular
Endocitosis
keywords: Trypanosoma cruzi
DeCS: Doença de Chagas
Espaço Intracelular
Endocitose
Mamíferos
Issue Date: 2015
Publisher: Public Library of Science
Citation: BATISTA, Cassiano Martin et al. Trypanosoma cruzi intracellular amastigotes isolated by nitrogen decompression are capable of endocytosis and cargo storage in reservosomes. PLoS ONE, v. 10, n. 6, p. 1-18, 2015.
DOI: 10.1371/journal.pone.0130165
ISSN: 1932-6203
Copyright: open access
Appears in Collections:PR - ICC - Artigos de Periódicos

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