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First published online 15 February 2005
doi: 10.1242/jcs.01677

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Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, C.C.S., Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil

View larger version (20K): [in a new window]   Fig. 3. Overexpression of functional chagasin in transfected T. cruzi. (A) Epimastigotes were washed twice in PBS and lysed in PBS containing 1% Triton X-100. The lysates (100 µg) were resolved by SDS-PAGE and submitted to western blot using anti-chagasin antiserum (1:1000). Anti-calreticulin monoclonal antibodies were used as a control for sample loading in the gels. (B) Inhibitory activity in transfected epimastigotes. Lysates (2.4 µg) were boiled for 20 minutes and the soluble fraction was collected after 10,000 g centrifugation. Equal volumes of the soluble fraction were incubated with papain (3.32 nM) in 50 mM Na2PO4, 100 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT for 15 minutes at room temperature and the residual activity was subsequently monitored by the hydrolysis of 5 µM CBZ-Phe-Arg-AMC in the same buffer containing 5% DMSO. As a control, papain was incubated for the same period in buffer and the peptidase activity was measured in the same conditions. (C) Chagasin overexpression leads to a reduction in CP activity. The peptidase activity contained in normalized epimastigote lysates (1 µg) was determined in 50 mM Na2HPO4, 200 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO at room temperature, using 5 µM CBZ-Phe-Arg-AMC as a substrate. Substrate hydrolysis was fully inhibited by 10 µM E-64 (data not shown), confirming that the activity detected corresponds to that of CPs. The graph shows the initial velocities for the substrate hydrolysis curves. The experiments were performed in triplicate and are represented as mean values with standard deviations (SD). The analysis of variance was performed using ANOVA and the asterisk indicates the scores that are statistically significant at P<0.05 (B) or P<0.001 (C).

View larger version (21K): [in a new window]   Fig. 6. Chagasin overexpression leads to reduced extracellular CP activity. The secretion products of epimastigotes were obtained by resuspending washed cells in PBS and maintaining them for 3 hours at 28°C. The supernatants were collected by centrifugation at 3000 g and filtered through 0.2 µm membranes. (A) The peptidase activity present in the supernatants were measured in 50 mM Na2HPO4, 200 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO using 5 µM CBZ-Phe-Arg-AMC. Substrate hydrolysis was fully inhibited by E-64 (not shown). (B) Detection of the cruzipain-chagasin complex in supernatants. Supernatants were diluted in SDS-ßME buffer, not boiled, resolved by SDS-PAGE, blotted onto nitrocellulose and probed with anti-chagasin antiserum. (C) Supernatants were boiled for 20 minutes centrifuged at 10,000 g and the inhibitory activity present in the soluble fraction was determined upon incubation with papain in 50 mM Na2HPO4, 100 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT for 15 minutes at room temperature. After incubation, the residual activity of papain was measured by the hydrolysis of CBZ-Phe-Arg-AMC. The graphs represent the initial velocities for the substrate hydrolysis curves. The experiments were performed in triplicates and are represented as the mean values with the standard deviations (SD). The analysis of variance was performed using ANOVA and the asterisks indicate the scores that are statistically significant at P<0.001 (A) and P<0.01 (C).

View larger version (11K): [in a new window]   Fig. 7. Chagasin overexpression affects parasite differentiation. Stationary-phase epimastigotes were inoculated in 1:5 dilution (vol:vol) in Grace's insect medium, pH 6.5, and cultivated at 28°C for 10 days. The proportion of trypomastigotes in the cultures was estimated by daily counting under the light microscope starting at day 5. The experiments were performed in triplicate and are represented as the mean values with standard deviations (SD). The graph is representative of three independent experiments. The rate of parasite differentiation was analysed using ANOVA and the asterisk indicates statistical significance at P<0.05.

View larger version (14K): [in a new window]   Fig. 11. Chagasin impairs mammalian-cell invasion by trypomastigotes. (A) Mammalian-host-cell invasion by WT tissue-culture trypomastigotes. Human primary culture of smooth-muscle cells plated on glass coverslips were incubated with T. cruzi tissue-culture trypomastigotes at a 5:1 parasite:host-cell ratio for 3 hours at 37°C in DMEM supplemented with 0.1% BSA in the presence of buffer or of recombinant chagasin at different concentrations. The number of intracellular parasites was estimated by counting under the light microscope. All the assays were performed in triplicate in three independent experiments and are represented as mean values with standard deviations (SD). The variance was analysed using ANOVA and the linear trend post-test. The analysis indicated that there is a significant systematic decrease of invasion (slope=–3.94), at P<0.0001. (B) Recombinant chagasin and E-64 inactivate host-cell CPs in living cells. Smooth-muscle cells grown to semiconfluence were incubated in DMEM plus 2% FCS for 1 hour at 37°C, with buffer alone, buffer containing 10 µM E-64 or different concentrations of recombinant chagasin. The cells were washed three times with HBSS and lysed in 100 mM sodium acetate, 150 mM NaCl, pH 5.5, 1% Triton X-100 and incubated for 10 minutes on ice. The soluble fraction was recovered by centrifugation at 10,000 g for 10 minutes and 0.25 µg ml–1 lysates were assayed for peptidase activity as described above. All the assays were performed in triplicate, in three independent experiments and are represented as mean values with standard deviations (SD). The variance was analysed using ANOVA and the linear trend post-test. The analysis indicated that there is a significant systematic decrease of activity (slope=–0.47) at P<0.0098. (C) T. cruzi tissue-culture trypomastigotes were obtained from infected LLCMK2 monolayers. Freshly released trypomastigotes were washed twice in HBSS and incubated with human smooth-muscle cultures at a 3:1 parasite:host-cell ratio in DMEM plus 0.1% BSA for 3 hours at 37°C. Extracellular parasites were removed and the cells were fixed and stained as described in A. All experiments were performed in triplicate. The graph is representative of three independent experiments. The analysis of variance was performed using ANOVA and the asterisk indicates the score with statistical significance at P<0.001. (D) Membrane-associated CPs restore the infectivity of trypomastigotes overexpressing chagasin. Tissue-culture trypomastigotes were washed twice in HBSS and incubated in this solution for 2 hours at 37°C to allow membrane shedding. The parasites were removed by centrifugation at 3000 g and the cell-free supernatant was filtered through a 0.22 µm pore-size membrane. The filtered sample was submitted to 100,000 g centrifugation for 1 hour at 4°C and the pellet (membranes) was resuspended to the original volume in HBSS plus 0.1% BSA. Tissue-culture trypomastigotes overexpressing chagasin (pCHAG) were incubated with smooth-muscle cells at a 15:1 parasite:host-cell ratio for 3 hours at 37°C in DMEM plus 0.1% BSA in the presence of HBSS supplemented with 2.5 mM DTT or in the presence of 100 µl membrane fraction recovered from WT trypomastigotes supplemented with 2.5 mM DTT. Where indicated, E-64 was added at a 10 µM final concentration. The variance was analysed using ANOVA and the asterisks indicate the scores showing statistical significance at P<0.01. (Inset) The peptidase activity present in the membrane fraction (100 µl) recovered from different trypomastigotes was assayed in 50 mM Na2PO4, 100 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO by the hydrolysis of 5 µM CBZ-Phe-Arg-AMC. Substrate hydrolysis was fully inhibited by E-64 (not shown). The graph represents initial velocities.

View larger version (17K): [in a new window]   Fig. 8. Chagasin overexpression partially protects T. cruzi epimastigotes against the toxicity of the synthetic CP inhibitor N-Pip-F-hF-VSPh. Epimastigotes were inoculated at 106 ml–1 in LIT medium containing 10% FCS and 0.5% DMSO in the presence of 2 µM, 4 µM, 6 µM or 8 µM N-Pip-F-hF-VSPh, and cultivated for 5 days at 28°C. The controls were cultivated solely in the presence of the DMSO diluent. (A) WT. (B) MOCK. (C) pCHAG. The experiments were performed in triplicate and are reported as mean values with standard deviations (SD). The graphs are representative of two independent experiments. The analyses of variance were performed using two-way ANOVA and the Bonferroni post-test at a significance level of 5%. The single asterisks show the scores that are statistically significant at P<0.05 and the triple asterisks show the scores that are statistically significant at P<0.01. In the WT and MOCK individual graphs, the variation in the growth rate in the presence of all drug concentrations were equally significant in relation to growth in the presence of DMSO. The variances in the growth rates of pCHAG were significant in relation to the control and among the various drug concentrations. The variance in the growth rates of pCHAG in the presence of 6 µM and 8 µM N-Pip-F-hF-VSPh was extremely significant (P<0.0001) in relation to WT and MOCK.

View larger version (112K): [in a new window]   Fig. 1. Cruzipain and chagasin co-localize to reservosomes and the Golgi apparatus of T. cruzi epimastigotes. Epimastigotes were fixed in 4% paraformaldehyde, adhered on poly-L-lysine-coated glass coverslips and permeabilized with PBS containing 1% NP-40. The parasites were incubated overnight at 4°C with anti-chagasin rabbit antiserum or mouse anti-cruzipain antiserum, followed by incubation with Alexa-543-conjugated anti-rabbit and FITC-conjugated anti-mouse antibodies. (A) Differential-interference-contrast image. (B) Anti-cruzipain labelling. (C) Anti-chagasin labelling. (D) Merged image of anti-cruzipain and anti-chagasin labelling. Arrows and arrowheads, respectively, indicate the posterior and anterior ends of the cell. Scale bar, 2 µm. Epimastigote cryosections were incubated with rabbit anti-chagasin antiserum or mouse anti-cruzipain antiserum at 1:100 and 1:500 dilutions. The sections were subsequently incubated with goat anti-rabbit IgG conjugated to 5 nm gold particles and goat anti-mouse IgG conjugated to 15 nm gold particles (at 1:100), respectively. (E) T. cruzi reservosomes. (F) A higher-magnification image of the interior of a reservosome showing cruzipain and chagasin in close proximity. (G) A reservosome (R) at high magnification, next to the Golgi complex (GC). Dark arrows indicate cruzipain staining, arrowheads indicate chagasin staining and white arrows point to chagasin inside cruzipain-free small vesicles. Scale bars, 0.1 µm (E), 0.4 µm (F) and 0.1 µm (G).

View larger version (13K): [in a new window]   Fig. 2. Detection of the cruzipain-chagasin molecular complex in vitro and in vivo. (A) The cruzipain-chagasin complex is stable upon treatment with SDS-ßME. Purified cruzipain and recombinant chagasin were incubated at equal concentrations in PBS, pH 7.2, for 20 minutes and subsequently diluted in different solutions: buffer alone (62.5 mM Tris-HCl pH 6.8, 10% glycerol) (lane 1); buffer containing 2% SDS; buffer containing 2% SDS and 5% ßME; and buffer containing 2% SDS and 5% ßME, and boiled for 5 minutes. The samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes and incubated with rabbit anti-chagasin antiserum (1:1000). (B) Free cruzipain is inactivated by SDS-ßME. The cruzipain-chagasin complex was formed as described in A and incubated in buffer containing 2% SDS and 5% ßME (lane 1) or cruzipain was diluted in the SDS-ßME buffer immediately before the addition of chagasin (lane 2). The samples were not boiled before loading in SDS-PAGE. Western blots with anti-chagasin antibodies were performed as described above. (C,D) Detection of the cruzipain-chagasin complex in living parasites. Epimastigotes (5x106) were washed twice in PBS and lysed directly by addition of a solution containing 2% SDS and 5% ßME. The samples (not boiled) were resolved by SDS-PAGE and submitted to western blot with anti-chagasin antibodies (C) or anti-cruzipain anti-serum (1:1000) (D).

View larger version (25K): [in a new window]   Fig. 4. Chagasin overexpression results in downregulation of soluble, but not of membrane-associated, cruzipain. In-vivo active-site labelling of CPs: epimastigotes were incubated in LIT medium containing 10 µM biotin-N-Pip-F-hF-VSPh (an irreversible CP inhibitor) for 1 hour at 28°C. The cells were washed twice in ice-cold PBS, lysed in this buffer by freeze-thaw and fractionated by 100,000 g centrifugation. The pellet was resuspended in PBS containing 1% Triton X-100 to the original volume. The samples were submitted to SDS-PAGE, transferred to nitrocellulose and the reactive bands visualized upon incubation with streptavidin/alkaline-phosphatase. The relative intensities of the reactive bands were estimated by scanning densitometry and normalised to the WT, and are indicated below each lane. (A) Soluble fraction. (B) Membrane fraction.

View larger version (34K): [in a new window]   Fig. 5. Epimastigotes overexpressing chagasin accumulate more of the cruzipain-chagasin complex. Epimastigotes (5x106) were lysed directly in the SDS-ßME buffer (not boiled) and submitted to a western blot. The high-molecular-weight complex was visualized using anti-chagasin antiserum.

View larger version (20K): [in a new window]   Fig. 9. CP and chagasin expression in different T. cruzi isolates. (A) Immunoblotting of epimastigote lysates probed with monoclonal antibody against cruzipain, monoclonal antibodies against the cathepsin-B-like enzyme of T. cruzi (TcCB) or monoclonal antibodies against calreticulin. (B) Detection of CP activity in epimastigote lysates (1 µg) in 50 mM Na2HPO4, 200 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO at room temperature using 5 µM CBZ-Phe-Arg-AMC as a substrate. Substrate hydrolysis was fully inhibited by E-64 (not shown). The experiments were performed in triplicate and are represented as mean values with standard deviations (SD). The variance was analysed using ANOVA and the asterisks indicate the scores that are statistically significant at P<0.001. (C) Immunoblotting of epimastigote lysates probed with anti-chagasin antibodies or with monoclonal antibodies against calreticulin. The relative intensities of the reactive bands were estimated by scanning densitometry normalized to Dm28c and are indicated below each lane.

View larger version (21K): [in a new window]   Fig. 10. Selectivity of the CP inhibitor N-Pip-F-hF-VSPh to cruzipain over TcCB. Epimastigote lysates were incubated with 10 µM biotin-N-Pip-F-hF-VSh (lane 2) or biotin-LVG-CHN2 (lane 1) in 50 mM Na2HPO4, 200 mM NaCl, 5 mM EDTA, pH 6.5, 5 mM DTT for 2 hours at room temperature. Reactions were stopped by the addition of SDS-PAGE sample buffer, boiled for 3 minutes and analysed by western blot upon incubation with phosphatase-conjugated streptavidin.

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