Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes.
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 prolife...
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4461355?pdf=render |
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| author | Cassiano Martin Batista Rafael Luis Kessler Iriane Eger Maurilio José Soares |
| author_facet | Cassiano Martin Batista Rafael Luis Kessler Iriane Eger Maurilio José Soares |
| author_sort | Cassiano Martin Batista |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T17:16:53Z |
| format | Article |
| id | doaj.art-35b1145bf3134fb699113ff616d6471e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T17:16:53Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-35b1145bf3134fb699113ff616d6471e2022-12-21T18:56:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01106e013016510.1371/journal.pone.0130165Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes.Cassiano Martin BatistaRafael Luis KesslerIriane EgerMaurilio José SoaresEpimastigote 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.http://europepmc.org/articles/PMC4461355?pdf=render |
| spellingShingle | Cassiano Martin Batista Rafael Luis Kessler Iriane Eger Maurilio José Soares Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. PLoS ONE |
| title | Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. |
| title_full | Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. |
| title_fullStr | Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. |
| title_full_unstemmed | Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. |
| title_short | Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. |
| title_sort | trypanosoma cruzi intracellular amastigotes isolated by nitrogen decompression are capable of endocytosis and cargo storage in reservosomes |
| url | http://europepmc.org/articles/PMC4461355?pdf=render |
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