Improved method for in vitro secondary amastigogenesis of Trypanosoma cruzi: morphometrical and molecular analysis of intermediate developmental forms

Abstract

Trypanosoma cruzi undergoes a biphasic life cycle that consists of four alternate developmental stages. In vitro conditions to obtain a synchronic transformation and efficient rates of pure intermediate forms (IFs), which are indispensable for further biochemical, biological, and molecular studies, have not been reported. In the present study, we established an improved method to obtain IFs from secondary amastigogenesis. During the transformation kinetics, we observed progressive decreases in the size of the parasite body, undulating membrane and flagellum that were concomitant with nucleus remodeling and kinetoplast displacement. In addition, a gradual reduction in parasite movement and acquisition of the amastigote-specific Ssp4 antigen were observed. Therefore, our results showed that the in vitro conditions used obtained large quantities of highly synchronous and pure IFs that were clearly distinguished by morphometrical and molecular analyses. Obtaining these IFs represents the first step towards an understanding of the molecular mechanisms involved in amastigogenesis.

Figure 1

Transformation kinetics from tissue-derived trypomastigote to amastigote in hgDMEM at pH 5 without FBS at 37°C: relative percentage of trypomastigotes (-♦-), IFs (-■-), and amastigotes (-▲-) analyzed in fresh preparations under the light microscope. The results are the average of 3 independent experiments.

Figure 2

Morphological analysis of the kinetics of extracellular differentiation from tissue-derived trypomastigotes (a) to amastigotes (h), in hgDMEM at pH 5 without FBS at 37°C: intermediate forms at 1 hour (b), 2 hours (c), 3 hours (d), 4 hours (e), 5 hours (f), and 6 hours (g) of transformation stained with Hema 3. Bar = 25 μm.

Figure 3

The shape and position of the nucleus and the kinetoplast identified the different IFs. The morphology and position of the nucleus and the kinetoplast were determined by indirect immunofluorescence and DAPI staining of trypomastigotes (a), IFs at 1 hour (b), 2 hours (c), 3 hours (d), 4 hours (e), 5 hours (f) and 6 hours (g) of transformation and amastigotes (h). Bar = 25 μm.

Figure 4

Highly synchronous morphologic changes are obtained during the in vitro transformation kinetics. Quantitative analysis of 2 independent experiments of extracellular differentiation from tissue-derived trypomastigotes into amastigotes stained by indirect immunofluorescence and DAPI after 1, 2, 3, 4, 5 and 6 hours of transformation. (a) Relative percentage of trypomastigotes (-♦-), IFs (-■-) and amastigotes (-▲-). (b) Three different IFs arbitrarily designated as IF1 (black), IF2 (grey) and IF3 (white) were observed from 1 to 5 hours of transformation. (c) Representative parasite of the corresponding IFs of panel b.

Figure 5

Secondary amastigogenesis is accompanied by the gradual acquisition of amastigote-specific Ssp4 surface glycoprotein. The localization of Ssp4 was determined by indirect immunofluorescence in trypomastigotes (a, b), IFs at 1 hour (c, d), 2 hours (e, f), 3 hours (g, h), 4 hours (i, j), 5 hours (k, l) and 6 hours (m, n) of transformation and in amastigotes (o, p). The right panels are the immunofluorescence image (b, d, f, h, j, l, n, p) of the corresponding light microscopy panels (a, c, e, g, i, k, m, o).

Figure 6

Analysis of resistance to complement-mediated lysis. The relative percentages of trypomastigotes (-♦-), amastigotes (-■-), IFs at 3 hours of transformation (-▲-) and epimastigotes (-●-) were determined after treatment with fresh human serum. The results are the average of 3 independent experiments.