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First published online 9 September 2003
doi: 10.1242/jcs.00756

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Inhibition of caspase activation and a requirement for NF-B function in the Toxoplasma gondii-mediated blockade of host apoptosis

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose St, Lexington, KY 40536, USA

View larger version (33K): [in a new window]   Fig. 6. Toxoplasma cannot block activated caspases. Uninfected cells were treated with either 150 nM STS (C) or 25 ng ml–1 TNF (A,C) to induce apoptosis. Cell extracts of these uninfected apoptotic (UA) cells were mixed with cell extracts of either uninfected non-apoptotic (UNA) cells or infected non-apoptotic (INA) cells in a range of concentrations [shown as UA:UNA (gray bars) or UA:INA (white bars) ratios]. The mixtures of cell extracts were measured for caspase activity using the appropriate fluorogenic substrate for caspase 3 (A), caspase 8 (B) and caspase 9 (C). The line represents an expected decrease of 10% that corresponds to the 10% decrease in apoptotic cell extract (A-C). Error bars represent the standard deviation from triplicate samples. (D) Predictions from this experiment are presented in a hypothetical model. Accordingly, the presence of a specific inhibitor in extracts from infected cells (UA:INA, white bars), would result in a non-linear decrease in caspase activity (dashed line). By contrast, the absence of this specific inhibitor in infected cell extracts, would result in a decrease in activity, reflecting the dilution of the apoptotic cell extract (solid line). The absence of inhibitor in UA:INA (white bars) would result in a profile identical to that found in UA:UNA (gray bars).

View larger version (68K): [in a new window]   Fig. 2. T.-gondii-infected 3T3 fibroblasts resist nuclear fragmentation and the loss of plasma membrane asymmetry following apoptotic stimulation. (A) Nuclear fragmentation following treatment of 3T3 fibroblasts was visualized using the TUNEL assay (a,e,i). TUNEL positive cells are apparent in both STS (25 nM) and TNF/CX (25 ng ml–1 / 10 µg ml–1) treated cells. Notably, cells infected with Toxoplama, visualized using an antibody against the surface antigen SAG1 (b,f,j), remain TUNEL negative. In addition, the changes in nuclear morphology are apparent using Hoechst dye (c,g,k). Nuclei of apoptotic cells (TUNEL positive) appear fragmented and condensed compared with those of infected cells. Images of the TUNEL assays, SAG1 and Hoechst were pseudo-colored green, red and blue, respectively, and merged using Adobe Photoshop (d,h,l). Infected cells bearing differing parasite loads are apparent by the juxtanuclear SAG1 labeling. The nuclei of these cells remain TUNEL negative. Scale bar, 10 µm. (B) Fluorescence-activated cell sorting (FACS) analysis following Annexin-V/FITC labeling of untreated uninfected and infected cells (red trace) and cells triggered to undergo apoptosis using STS (300 nM for 5 hours) (green trace). Treatment of uninfected 3T3 cells with STS triggers apoptosis and the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, an event recognized by the increased binding of Annexin-V/FITC, resulting in the appearance of a new peak (Uninfected, green trace). By contrast, identically treated infected cells exhibit considerable resistance to Annexin-V binding, indicating an inhibition of phosphatidylserine translocation. Necrotic cells in the experiment were identified using propidium iodide staining and excluded from the analysis (data not shown).

View larger version (109K): [in a new window]   Fig. 1. T.-gondii-infected 3T3 cells do not exhibit features characteristic of apoptosis. Uninfected and infected 3T3 fibroblasts were left untreated (No treatment) or stimulated to undergo apoptosis using 10 nM staurosporine (STS) or 10 ng ml–1 TNF in the presence of cycloheximide (TNF/CX) for 8 hours. The cells were processed for electron microscopy. STS-treated uninfected cells (B) exhibit characteristic signs of apoptosis including chromatin condensation (arrowheads), vacuolation and the disorganization of cellular architecture. Following treatment with TNF/CX, uninfected wild-type fibroblasts exhibit extensive chromatin condensation and cellular disruption. By contrast, T.-gondii-infected cells (bottom) fail to exhibit the classic signs of apoptosis and display normal nuclear morphology and cytoplasmic organization (E,F). In the presented images, cellular debris is evident, presumably from an uninfected apoptotic cell(s). Scale bars, 2 µm.

View larger version (36K): [in a new window]   Fig. 3. Inhibition of caspase 3 activity in T.-gondii-infected cells. The effect of T. gondii infection (20 hours) on the activity of caspase 3 following treatment with STS (A,C) or TNF (B,D) for 8 hours was examined over a broad concentration range. Caspase 3 activity was detected using the intrinsic substrates 2-spectrin (A,B, Spectrin) or PARP (A,B, PARP). Caspase 3 activity can be detected in immunoblot analysis by the generation of the specific 120 kDa spectrin fragment (A,B, Spectrin, arrowhead) and the 89 kDa PARP fragment (A,B, PARP, arrowhead). Uninfected cells exhibit these characteristic bands with increasing concentrations of the apoptogenic trigger. By contrast, T.-gondii-infected cells exhibit a significant inhibition of caspase 3 activity. Low levels of activity in the infected sample are attributable to uninfected cells or, in the case of STS, probable toxicity towards the parasite. Caspase 3 activity was also measured in STS (C) and TNF (D) treated cells using the direct cleavage of a fluorescent substrate (DEVD-MCA). Activity was determined based on the fluorescence caused by the release of MCA. Based on the fluorometric analysis, infected cells (white bars) exhibit considerably lower levels of activity than uninfected cells (gray bars) across a broad range of concentrations of STS (C) and TNF (D). In the case of induction of apoptosis by TNF, the addition of cycloheximide is noted by a plus (+) sign. Error bars represent standard deviations using triplicate samples.

View larger version (42K): [in a new window]   Fig. 4. Inhibition of caspase 3 is due to a block of caspase activation. Activation of caspase 3 was detected by immunoblot (A,B) and immunofluorescence (C) analysis. Upon activation by upstream caspases, caspase 3 is proteolytically cleaved from the 32 kDa inactive zymogen to a 17-20 kDa and 11kDa active form. Following stimulation of apoptosis using STS for 8 hours (A), the active form of caspase 3 is detected as an 18 kDa band in uninfected cells (A, Uninfected, arrowhead). This conversion is blocked in identically treated T.-gondii-infected cells (A, Infected) where the 18 kDa band is not actively generated. Identical results are obtained using TNF-mediated apoptosis in the presence of cycloheximide (B). (C) Immunofluorescence microscopy of STS-treated (150 nM for 6 hours) uninfected and infected 3T3 fibroblasts using an antibody specific for the active form of caspase 3 (a,d). Toxoplasma-infected cells were detected using an antibody against the secreted antigen GRA3 (b,e). STS treatment causes morphological changes in both infected and uninfected cells (c,f). Notably, uninfected cells (lacking GRA3 labeling) primarily contain activated caspase 3 (c,f, black boxes), whereas those that are infected rarely label with the antibody against activated caspase 3 (c,f, white boxes). In the presented experiment, 15% of uninfected cells and 4% of infected cells exhibited staining with antibody against activated caspase 3 above the background level. Scale bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 5. Activity of initiator caspases is inhibited in Toxoplasma-infected cells. Caspase 8 (A,B) and caspase 9 (C,D) activity were measured following treatment with either STS (A,C) or TNF (B,D) using the direct cleavage of the fluorescent substrates LEHD-MCA and IETD-MCA, respectively. Activity was determined based on the fluorescence caused by the release of MCA. Based on the fluorometric analysis, infected cells (white bars) exhibit considerably lower levels of activity than uninfected cells (gray bars) for both caspase 8 (A,B) and caspase 9 (C,D) across a broad range of concentrations of STS (A,C) and TNF (B,D). In the case of induction of apoptosis by TNF, the addition of cycloheximide is noted by a plus (+) sign. STS-treated samples were harvested 4 hours after apoptotic induction. Error bars represent standard deviations using triplicate samples.

View larger version (34K): [in a new window]   Fig. 7. Toxoplasma infection fails to block the activity and activation of caspase 3 in p65–/– fibroblasts. The effect of T. gondii infection (20 hours) on the activity of caspase 3 following treatment with STS (A,B) or TNF (A,C) for 8 hours was examined over a broad concentration range. Caspase 3 activity was detected using the intrinsic substrate PARP (A, PARP). Unlike wild-type fibroblasts, infected p65–/– fibroblasts exhibit an identical level of the cleaved 89 kDa form as uninfected cells under both STS (top) and TNF (middle) treatment. The complete cleavage of PARP in CX/TNF samples reflects their hypersensitivity to TNF-mediated apoptosis. Identical results were obtained in the absence of CX (data not shown). The cleavage of PARP correlates with the activation of caspase 3, which is not inhibited in Toxoplasma-infected p65–/– fibroblasts (A, bottom). Immunoblots in the middle and bottom panels were performed on the same source material. Activity of caspase 3 measured using the fluorescent substrate (DEVD-MCA) cleavage assay supports the immunoblot data, indicating that no protection is afforded by T. gondii infection (white bars) compared with uninfected p65–/– cells (gray bars) across a broad concentration range following STS treatment (B). Similarly, TNF treatment triggers a dramatic activation of caspase 3 (notice scale for relative fluorescence) in both infected (white bars) and uninfected (gray bars) cells (C). This is consistent with the complete cleavage of PARP observed by immunoblot analysis. The apparent partial protection observed with infected cells at lower concentrations of TNF might indicate that there might be an alternate mechanism of protection independent of NF-B function. Alternatively, death might be more rapid in infected cells with highly activated caspase 3, resulting in the loss and lysis of a significant population prior to harvesting. In the case of induction of apoptosis by TNF, the addition of cycloheximide is noted by a plus (+) sign. Error bars represent standard deviations using triplicate samples.

View larger version (111K): [in a new window]   Fig. 8. T. gondii infection of p65–/– cells fails to protect them from apoptosis. Uninfected and infected p65–/– fibroblasts were left untreated (No treatment) or stimulated to undergo apoptosis using 10 nM staurosporine (STS) or 10 ng ml–1 TNF in the presence of cycloheximide (TNF/CX) for 8 hours. Uninfected cells treated with STS exhibit the classic signs of apoptosis, including nuclear fragmentation, vacuolation and a generalized disorganization of cellular architecture (B). In contrast to wild-type fibroblasts, these features are also observed in STS-treated T.-gondii-infected fibroblasts (E). Similarly, treatment of p65–/– cells with TNF results in the profound nuclear condensation and reorganization of cellular architecture in both uninfected (C) and T.-gondii-infected cells (F). Scale bars, 2 µm.

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