Fig 1.
Trypanosoma cruzi intracellular amastigotes isolated by nitrogen decompression have normal shape, high levels of Ssp4, and cruzipain labeling in posterior organelles.
(A) Differential interference contrast (DIC) image of the fraction of isolated intracellular amastigotes obtained after nitrogen decompression and differential centrifugation. Note that most cells have the typical amastigote shape. Scale bar, 10 μm. (B) Flow cytometry analysis of axenic and isolated intracellular amastigotes, culture epimastigotes and in vitro-derived trypomastigotes labeled with an antibody against Ssp4, a specific amastigote marker. Trypomastigotes and epimastigotes had low levels of fluorescence signal (possibly due to auto-fluorescence), while axenic and intracellular amastigotes showed higher fluorescence intensity. (C) Immunofluorescence microscopy of isolated intracellular amastigotes labeled with an anti-Ssp4 antiserum (green, in a-d), or with the anti-cruzipain monoclonal antibody CZP-315.D9 (green, in e-h). The nucleus (arrow) and the kinetoplast (arrowhead) are stained blue with Hoechst 33342, and parasite morphology was visualized by DIC. While Ssp4 localizes to the cell surface, the cruzipain signal is found specifically in lysosome related organelles posterior to the nucleus. Scale bars, 5 μm.
Fig 2.
Western blotting analysis of transferrin-AlexaFluor 633 uptake in isolated Trypanosoma cruzi intracellular amastigotes.
(A) Detection of holo-transferrin (using anti-transferrin goat antiserum) in total protein extracts of isolated intracellular amastigotes and epimastigotes (positive control for endocytosis) endocytosis assays at different temperatures. The panel on the left shows the Ponceau-stained membrane used for transferrin immuno-blotting analysis (on the right). The protein band with approximately 80 kDa corresponds to transferrin. Protein extract from isolated intracellular amastigotes incubated with transferrin at 37°C showed a strong band, indicating transferrin internalization. TcGAPDH (~40 kDa) was used as a loading control. (B) Ratios between the integrated densities of the transferrin band at 28 or 37°C and 4°C (Y-axis), based on measurements performed using ImageJ.
Fig 3.
Flow cytometry analysis of transferrin-AlexaFluor 633 endocytosis by amastigotes and epimastigotes of Trypanosoma cruzi.
(A) Flow cytometry histograms of parasites incubated with transferrin at different temperatures, and then treated with acetic acid, or left untreated. Epimastigotes were used as positive control, and negative control parasites (black) were incubated in medium without labeled transferrin. These data show that isolated intracellular amastigotes internalize transferrin at 37°C. (B) Normalized medians (stained/unstained control) of the fluorescence peaks. Note the low endocytosis levels in axenic amastigotes (N = 3). ***p<0.001. a, acetic acid treatment.
Fig 4.
Fluorescence microscopy analysis of transferrin-AlexaFluor 633 endocytosis by Trypanosoma cruzi epimastigotes.
Cells were allowed to ingest transferrin and were then labeled with an anti-transferrin antibody (in red) and with the anti-cruzipain mAb CZP-315.D9 (CZP; in green). At 4°C no co-localization was observed (A), while transferrin and cruzipain co-localized at the posterior region of the cell after incubation at 28°C (B), resulting in yellow staining of the reservosomes. The nucleus (arrow) and kinetoplast (arrowhead) are stained with Hoechst 33342 (in blue). DIC, differential interference contrast. Scale bars, 5 μm.
Fig 5.
Fluorescence microscopy analysis of transferrin-AlexaFluor 633 endocytosis by isolated intracellular amastigotes of Trypanosoma cruzi.
Cells were allowed to ingest transferrin-AlexaFluor 633 and were then incubated with anti-transferrin antibody (in red) and anti-cruzipain mAb (CZP-315.D9; in green). (A-B) Isolated intracellular amastigotes incubated without transferrin at 4°C or 37°C, showed no transferrin labeling (negative control), while cruzipain was located at the posterior region of the cell. (C) Isolated intracellular amastigotes incubated with transferrin at 4°C. No co-localization with cruzipain was observed. (D) Isolated intracellular amastigotes incubated with transferrin at 37°C. Co-localization with cruzipain was observed at the posterior region of the cell. (E-F) Isolated intracellular amastigotes incubated with transferrin at 4°C and 37°C and then subjected to acetic acid treatment. No co-localization of transferrin with cruzipain was observed at 4°C (E), while co-localization with cruzipain occurred at the posterior region at 37°C (F). The nucleus (n) and the kinetoplast (k) were stained with Hoechst 33342 (in blue). DIC, differential interference contrast. Scale bars, 2.5 μm.
Fig 6.
Flow cytometry analysis of albumin-AlexaFluor 488 endocytosis in T. cruzi by isolated intracellular amastigotes.
(A) Flow cytometry histograms of cells incubated with labeled albumin at different temperatures. Epimastigotes were used as positive control, and negative control parasites (black) were incubated in medium without labeled albumin. These data show that isolated intracellular amastigotes internalize low amounts of albumin at 37°C. (B) Normalized medians of the fluorescence peaks, relative to the negative control (N = 3). ***p<0.001.
Fig 7.
Fluorescence microscopy analysis of albumin-AlexaFluor 488 endocytosis by isolated intracellular amastigotes of T. cruzi.
Cells were allowed to ingest albumin-AlexaFluor 488 (in green) and were then labeled with the anti-cruzipain mAb CZP-315.D9 (in red). In epimastigotes (A) and isolated intracellular amastigotes (C) incubated at 4°C, we did not observe co-localization of albumin (at the anterior end of the cell, nearest to the kinetoplast—k) and cruzipain (at the posterior end of the cell, nearest to the nucleus—n). In contrast, in epimastigotes (B) and isolated intracellular amastigotes (D) incubated at endocytosis-permissive temperatures (28°C or 37°C, respectively), we detected co-localization of albumin and cruzipain at the posterior region of the cell. The nucleus (n) and kinetoplast (k) are stained with Hoechst 33342 (in blue). DIC, differential interference contrast. Scale bars, 5 μm.
Fig 8.
Schematic model of transferrin and albumin uptake by isolated Trypanosoma cruzi intracellular amastigotes.
As in epimastigotes, transferrin uptake by amastigotes occurs mainly through the cytostome/cytopharynx complex (Cy). After internalization, transferrin is directed to the reservosomes (R), where it is stored together with lysosomal proteases such as cruzipain (Cz), the major cysteine protease of this parasite. Albumin (A) is internalized via the flagellar pocket region, but albumin endocytic activity is low. The destination of ingested macromolecules is the reservosomes.