Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis

Abstract

Background: The transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation.

Methods: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression.

Results: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. In intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region.

Conclusions: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.

Figure 1

Expression of GP82 and GP90 at different time-points during T. cruzi metacyclogenesis. (A) Relative number of epimastigotes (blue), intermediate forms (green) and metacyclic trypomastigotes (red) in analyzed samples. Numbers are derived from three independent experiments where 200 cells were analyzed. A representative drawing of each parasite form is shown on the right. (B) Quantitative real-time PCR for determination of transcript levels. Means and standard deviations are derived from three independent experiments. The difference between epimastigotes and other forms was significant (P <0.05). (C) SDS-PAGE followed by Western blot showing the presence of GP82 and GP90 at 24 and 48 h. Total protein extract from 2.5 × 10⁶ parasites were used and anti-α-tubulin mAb was used as loading control. Experiments shown were carried out three times with similar results. (D) Live or permeabilized parasites were reacted with mAb 3F6 or 1G7 and processed for flow cytometry analysis. Values represent fluorescence means and standard deviation of independent experiments that were normalized by the fluorescence of parasites incubated only with secondary antibody. The difference between live and permeabilized parasites was significant for 24 and 48 h samples (P <0.05) and not significant (ns) for epimastigotes and metacyclic forms.

Figure 2

Differential cellular localization of GP82 and GP90 in parasites undergoing metacyclogenesis. Immunofluorescence was performed using exponentially growing epimastigotes, parasites attached to culture flasks at 48 h after nutritional stress and metacyclic trypomastigotes purified by DEAE-cellulose. Cells were fixed, permeabilized with 0.5% saponin, reacted with mAb 1G7 (A) or 3F6 (B) and incubated with secondary antibody Alexa Fluor 488. DAPI was used to stain nucleus (N) and kinetoplast (K). Scale bar = 10 μm.

Figure 3

Colocalization of GP82 with cruzipain during metacyclogenesis. Immunofluorescence showing intermediate forms attached to culture flasks at 48 h (A-B) and late intermediate forms and fully differentiated metacyclic forms in culture supernatant (C-D) at 48 h after nutritional stress. Cells were fixed, permeabilized with 0.5% saponin and submitted to immunofluorescence using mAb 3F6 and rabbit polyclonal antibody against T. cruzi cruzipain. DAPI was used to stain nucleus (N) and kinetoplast (K). Scale bar = 10 μm.

Figure 4

Schematic representation of GP82 and GP90 pathways toward the cell surface during differentiation. Intermediate forms in which these proteins start to be expressed are represented. GP90 (red squares) mRNAs sequences encode an N-terminal signal peptide and a C-terminal signal anchor driving polypeptide through the ER-Golgi secretory pathway (1-2) to be glycosylated and receive GPI anchor. GP90 proteins leave the Golgi complex in secretory vesicles that fuse with the flagellar pocket membrane (3) and are distributed along the parasite plasma membrane (4). GP82 (green triangles) mRNAs also encode N-terminal and C-terminal signal sequences that drive polypeptide through the ER-Golgi pathway (1-2) to receive post-translational modifications (glycosylation and GPI anchor). However, instead of being addressed straight to the plasma membrane via flagellar pocket, GP82 leave the Golgi complex in vesicles that concentrate in lysosome-related organelles (LROs) (3). Further in the differentiation process, sorting mechanisms occurring at LROs and organelle repositioning allow vesicles carrying GP82 to fuse with the flagellar pocket membrane (4) and distribute the protein along the plasma membrane (5). N: nucleus, K: kinetoplast.