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Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression

Department of Bacteriology, Georg-August-University Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany

View larger version (64K): [in a new window]   Fig. 1. T. gondii-infected HL-60 and U937 cells are partially protected from fragmentation of genomic DNA induced by pro-apoptotic stimuli. Human-derived cells were infected at parasite to host ratios of 10:1 (+) and 30:1 (++) or were left uninfected. After 30 minutes, HL-60 and U937 cells were treated with 5 µg/ml actinomycin D or 40 ng/ml TNF- in combination with 2 µg/ml cycloheximide, respectively. Eight hours after infection, genomic DNA was analysed by agarose gel electrophoresis and ethidium bromide staining. DNA molecular weight markers (100 bp ladder) were separated in parallel. Signal intensities of bands corresponding to fragmentated genomic DNA were quantified by densitometry. Bars represent the relative intensities of representative bands, the intensities in uninfected HL-60 and U937 cells after treatment with pro-apoptotic stimuli were defined as 100%.

View larger version (62K): [in a new window]   Fig. 2. T. gondii downregulates activation of caspases 3 and 9 after induction of apoptosis in human-derived cell lines. HL-60 and U937 cells were infected at a parasite to host ratio of 30:1 and were 30 minutes later treated with actinomycin D or TNF- in combination with cycloheximide as indicated. Eight hours after infection, antigenic extracts were prepared from equal numbers of cells and were separated by standard SDS-PAGE. After transfer to nitrocellulose membranes, caspase 3 (A), caspase 9 (B) and caspase 8 (C) were visualized by immunostaining using enhanced chemiluminescence detection. T. gondii-induced alterations of caspase activation were quantified densitometrically by determining the levels of the active subunits (caspase 3) or the levels of the inactive proforms (caspases 8 and 9) (D). Bars represent the relative changes in caspase levels after parasitic infection of untreated cells (open bars) or those treated with pro-apoptotic stimuli (closed bars) compared with levels in uninfected cells. Horizontal dashed lines indicate an unchanged protein level after parasitic infection compared with levels in uninfected controls.

View larger version (53K): [in a new window]   Fig. 3. Expression of PARP as well as cleavage of nuclear target proteins during apoptosis in human-derived cell lines are decreased by T. gondii. After infection of HL-60 and U937 cells at a parasite to host ratio of 30:1, infected and uninfected control cells were treated with 5 µg/ml actinomycin D or 40 ng/ml TNF- in combination with 2 µg/ml cycloheximide as indicated. Eight hours after infection, antigenic lysates were prepared and separated by SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes and PARP (A) and PKC (B) were detected by immunostaining using enhanced chemiluminescence detection.

View larger version (44K): [in a new window]   Fig. 4. RT-PCR analyses of PARP transcript levels in T. gondii-infected and uninfected human-derived cell lines. After infection of HL-60 and U937 cells at a parasite to host ratio of 30:1, infected and uninfected control cells were treated with 5 µg/ml actinomycin D or 40 ng/ml TNF- plus 2 µg/ml cycloheximide or left untreated. Eight hours after infection, total RNA was isolated and PARP and ß-actin transcripts were reverse transcribed and amplified using an one-step RT-PCR protocol. Amplified mRNAs, a negative control without RNA (con), and a 100 bp ladder (M) were separated by agarose gel electrophoresis and visualized by ethidium bromide staining (A). Band intensities were quantified by densitometry. Bars represent the relative amount of amplified PARP mRNA normalized against ß-actin in the same sample (PARP/ß-actin x100), the amount of amplified PARP mRNA from untreated, uninfected control cells were defined as 100% (B). As a control, levels of PARP protein were determined in parallel by immunoblotting as described in Fig. 4 legend (C).

View larger version (55K): [in a new window]   Fig. 5. Apoptosis-associated cytochrome c-release from mitochondria into the cytosol of human-derived HL-60 cells is inhibited by T. gondii. HL-60 cells were infected at a parasite to host ratio of 30:1 and were then treated with 5 µg/ml actinomycin D for 8 hours. Equal numbers of cells per treatment were fractionated into digitonin-soluble and -insoluble extracts, containing cytosolic and mitochondrial proteins, respectively. Lysates were separated by SDS-PAGE and were then transferred to nitrocellulose membranes. Immobilized proteins were probed with a cytochrome c-specific (A) or a cytochrome c oxidase (COX) subunit IV-specific (B) monoclonal antibody and an appropriate secondary antibody using enhanced chemiluminescence detection. COX served as a marker for mitochondrial contamination of the digitonin-soluble extracts.

View larger version (56K): [in a new window]   Fig. 6. Intracellular distribution of cytochrome c in apoptotic and non-apoptotic human-derived HL-60 cells after infection with T. gondii. Cells were infected at a parasite to host ratio of 30:1 and were then treated with 5 µg/ml actinomycin D for 8 hours. The subcellular distribution of cytochrome c was determined by triple immunofluorescence staining and confocal microscopy. After fixation and permeabilization, cell preparations were stained using fluorescein-labelled dUTP to visualize DNA strand breaks (green fluorescence), a cytochrome c-specific monoclonal antibody and Cy3-conjugated secondary antibody (red fluorescence), and a Toxoplasma-specific antiserum and Cy5-conjugated secondary antibody (blue fluorescence).(A) Single optical sections from representative cells of the indicated treatments are shown. In non-apoptotic cells (i.e. those without signs of DNA strand breaks), cytochrome c was granularly distributed indicating a mitochondrial localization (thick arrows), while this molecule was homogenously distributed in apoptotic cells indicating translocation into the cytoplasm (arrowheads). Parasite-positive cells showed no signs of apoptosis and this was correlated with a granular (i.e. mitochondrial) distribution of cytochrome c (thin arrows). (B) 10 optical sections for each cell preparation were taken at intervals of 0.5 µm and were superimposed. A fluorescence intensity profile of the cytochrome c labelling was determined for selected cells as indicated by the straight line in the overlay micrographs. The lowercase letters below the intensity profiles refer to the cells or parts thereof for which the subcellular distribution of cytochrome c has been determined.

View larger version (45K): [in a new window]   Fig. 7. T. gondii upregulates protein levels of Mcl-1, but not Bcl-2, in human-derived cell lines. Thirty minutes after infection at a parasite to host ratio of 30:1, HL-60 and U937 cells were treated for 8 hours with 5 µg/ml actinomycin D or 40 ng/ml TNF- in combination with 2 µg/ml cycloheximide, respectively. Antigenic lysates were then separated by SDS-PAGE under reducing conditions, proteins transferred to nitrocellulose membranes and Bcl-2 (A) and Mcl-1 (B) were detected by immunostaining using enhanced chemiluminescence detection. T. gondii-induced alterations of Bcl-2 and Mcl-1 protein levels were quantified densitometrically (C). Bars represent the relative changes in protein levels after parasitic infection of untreated cells (open bars) or those treated with pro-apoptotic stimuli (closed bars) compared with levels in uninfected cells. Horizontal dashed lines indicate an unchanged protein level after parasitic infection compared with levels in uninfected controls.