Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation

Abstract

Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.

Figure 1

Recombinant expression of T. cruzi metacaspases in bacteria. (a) Schematic representation of full-length, active site mutants and truncated versions of TcMCA3 and TcMCA5 in the bacterial expression vector pBAD24. Metacaspases were tagged at the N and C termini with 6 × His-HA and 3 × Flag, respectively. (b) Cleavage of substrate Ac-VRPR-AMC by full-length, active site mutants and truncated versions of purified recombinant TcMCA3 and TcMCA5 produced in E. coli measured by fluorometric assay as described in Materials and Methods. Activity is expressed as the fold increase relative to Ac-VRPR-AMC hydrolysis generated by mock (empty vector) purification. Means and S.D. from four independent experiments are indicated. Differences observed between values were statistically significant (Student's t-test). ^*P<0.05 and ^***P<0.001. (c) Time course of substrate hydrolysis (150 μM Ac-VRPR-AMC) by 20 μg of purified recombinant protein (TcMCA3, TcMCA5 or TcMCA5ΔCt). Additions of 10 mM calcium or EDTA are indicated by arrows. (d) Western blot analysis of the reaction mixtures after the activity assays using anti-HA, anti-Flag, anti-TcMCA3 or anti-TcMCA5 antibodies. (e) Time-course experiment of recombinant proteins incubated with calcium and analyzed by SDS-PAGE, followed by Coomassie staining. Results are representative of at least three independent experiments

Figure 2

Inducible expression of metacaspases in epimastigotes. (a) Schematic representation of metacaspase constructs in the T. cruzi tetracycline-inducible vector pTcINDEX. (b) Cell lines harbouring transgenes encoding TcMCA3 active (C223) or inactive (C223A); TcMCA5 active (C201) or inactive (C201A) and TcMCA5ΔCt active (C201) or inactive (C201A) were grown in the absence (−) or presence (+) of tetracycline (Tet) for 60 h before cell lysates (10⁷ parasites per lane) were prepared and analyzed by western blot with anti-Flag or anti-HA antibodies. (c) Growth curve of wild-type (pLew13) epimastigotes and cell lines described in (b). Cells were grown in the presence (closed circles, continuous line) or absence (open circles, dashed line) of tetracycline and counted daily during 5 days. Results are representative of three independent experiments

Figure 3

Overexpression of full-length TcMCA3, but not its active site mutant, arrests cell cycle in G1/S transition. (a) FACS analysis of the cell cycle progression of synchronized TcMCA3 transgenic epimastigotes in the absence (upper row) or in the presence (middle row) of tetracycline. A similar analysis was performed for the active site mutant-induced cell line (lower row). (b) Uninduced (−) or induced (+) cultures of TcMCA3 were analyzed by western blot (10⁷ parasites per lane) at different time points after HU removal (0, 6, 12 and 18 h) using anti-Flag antibodies. For TcMCA3^C223A, only the induced cultures are shown

Figure 4

Metacaspase-3 expression levels correlate with cell cycle arrest. (a) Schematic representation of the fusion protein used as a fluorescent reporter of TcMCA3 expression. (b) TcMCA3-eGFP transgenic epimastigotes were grown in the absence (−) or presence (+) of tetracycline for 60 h before cell lysates (10⁷ parasites per lane) were prepared and analyzed by western blot with anti-GFP antibody. (c) Growth curve of TcMCA3-eGFP cell line in the presence (closed circles, continuous line) or absence (open circles, dashed line) of tetracycline. (d) Flow cytometry analysis of the cell cycle progression of synchronized TcMCA3-eGFP epimastigotes in the absence (upper row) or in the presence (lower row) of tetracycline. (e) Bivariate dot plots showing the distribution of green fluorescence on the y axis (TcMCA3-eGFP) versus the red fluorescence of PI staining (representing DNA content) on the x axis. The empty arrowheads depict cells with low TcMCA3-eGFP levels that can progress through the cell cycle. The filled arrowhead shows that cells with a high content of TcMCA3-eGFP remain arrested in the DNA synthesis phase

Figure 5

Metacaspase-5 lacking the C-terminal extension promotes apoptosis-like cell death. (a) FACS analysis of cell size (FSC-height) and DNA content (PI) of epimastigotes. Cell lines harbouring transgenes encoding TcMCA5ΔCt active (C223) or inactive (C223A) grown in the absence (−) or presence (+) of tetracycline. The arrow indicates the appearance of hypodiploid cells in the TcMCA5ΔCt induced culture. (b) Cell lines harbouring transgenes encoding TcMCA5 active (C201) or inactive (C201A) were grown in the presence of tetracycline for 72 h before cell lysates (10⁷ parasites per lane) were prepared and analyzed by western blot with anti-TcMCA5 or anti-TcMCA5ΔCt polyclonal antibodies

Figure 6

Metacaspase-3 activity protects epimastigotes from cell death, stimulates differentiation to metacyclics and affects replication inside the mammalian host. (a) Schematic representation of the life cycle of T. cruzi. The parasite has a complex life cycle, with four major stages: in the insect vector a replicative stage, the epimastigote and a non-replicative one, the infective metacyclic trypomastigote, are present; in the mammalian host, an obligate intracellular replicative form, the amastigote and a non-replicative one, invasive for host cells, the bloodstream trypomastigote. These forms differ in size, subcellular organization, in antigenic and some metabolic properties. (b) Epimastigotes were grown for 2 weeks to stationary phase, induced or not with tetracycline and incubated for 1 or 3 weeks more (early and late stationary phase). Histograms show PI uptake in uninduced (empty) or induced (filled) TcMCA3 and TcMCA3^C223A cell lines. (c) Metacyclogenesis rate in uninduced (−) or induced (+) cell lines harbouring transgenes encoding TcMCA3, TcMCA3^C223A was evaluated as described in Materials and Methods. Numbers indicate that metacyclogenesis increase relative to the corresponding uninduced control. Means and S.D. from at least three independent experiments are indicated. Differences observed between values were statistically significant (Student's t-test). ^***P<0.001. (d) The number of trypomastigotes released after 7 days of infection was evaluated for the same cell lines than in (b). Numbers indicate decrease in trypomastigote release relative to the corresponding uninduced control. Means and S.D. from three independent experiments are indicated. Differences observed between values resulted statistically significant (Student's t-test). ^**P<0.01. (e) Evaluation of intracellular amastigotes after 5 days of infection. DIC, DAPI anti-Flag and merged images (DAPI in blue and FLAG in green) of infected Vero cells with induced (+Tet) or uninduced (−Tet) active or inactive TcMCA3 forms are shown. Bar scales represent 10 μm