Apoptosis of murine and human macrophages induced by group B Streptococcus agalactiae (GBS) is likely an important virulence mechanism that is used by the bacteria to suppress the host immune response and to persist at sites of infection. The mechanisms by which GBS induces apoptosis are, however, largely unknown. In this study, we report that in murine macrophages GBS induces unique changes in the regulation and localization of the apoptotic regulators Bad, 14-3-3, and Omi/high-temperature requirement A2 and leads to the release of cytochrome c and the activation of caspase-9 and caspase-3. Furthermore, inhibition of caspase-3 impaired GBS-induced apoptosis of macrophages. The ability to modulate the activity of effector caspases may therefore represent an unexploited avenue for therapeutic intervention in GBS infections.

Apoptosis-mediated elimination of macrophages plays an important role in microbial pathogenesis and exerts unique changes on the host immune responses and on the persistence of pathogens in many dissimilar infections (1). Although some bacteria such as Chlamydia depend upon macrophages as a niche for survival and growth, the elimination of these infected cells by apoptosis represents an ideal means of simultaneously preventing pathological inflammatory responses while also diminishing the Ab- and neutrophil-protective cellular environment of the macrophage (2). In contrast, other bacteria have evolved to take advantage of apoptotic pathways to eliminate the very cells responsible for their clearance. For example, apoptosis of macrophages induced by bacteria such as Yersinia appears to dampen the immune response during infection, suppresses antimicrobial activity, and limits inflammation (3, 4). Thus, triggering macrophage apoptosis by extracellular bacteria such as Streptococci is believed to promote bacterial survival and dissemination and may tilt the balance of the host-microbial interaction to facilitate bacterial persistence.

Group B Streptococcus agalactiae (GBS) 3 is a major pathogen of neonates and in adults with underlying chronic disease (5). The organism asymptomatically colonizes up to 42% of pregnant women, and vertical transmission to infants occurs in up to 72% of these pregnancies (5). Respiratory colonization of neonates at birth leads to a lower respiratory tract infection, and alveolar macrophages are considered as important early effector cells against GBS in the lung. Opsonin-independent defenses appear critical in the early resistance of infants to GBS infection, since suboptimal levels of anti-GBS capsular polysaccharide Ab are typical of newborns. Neonates are also deficient in serum complement compared with adults. Following nonopsonic phagocytosis of GBS by macrophages (6, 7), GBS persist within macrophages (8, 9) and can induce apoptosis in host cells (10, 11, 12). Macrophage-derived NO contributes to GBS-induced apoptosis (13) and GBS β-hemolysin contributes to the apoptotic response triggered by GBS (10, 12), although additional GBS factors have also been indicated (14). GBS-induced macrophage apoptosis appears to represent an important strategy used by the bacterium that limits inflammation during infection and may facilitate bacterial escape from normal host immune defenses.

Critical regulators of apoptosis include the caspase family of cysteine-directed proteases and members of the Bcl-2 family of apoptotic regulators. These factors control cell death throughout the initiation, signal transduction, and execution phases of apoptosis. Caspases such as caspase-9 and caspase-8 initiate cell death, whereas others, such as caspase-3, are terminal effectors that commit cells to death once they are activated. Many Bcl-2 family proteins, in contrast, reside at the outer mitochondrial membrane, where they promote cell survival by binding to and suppressing the activities of functionally competing proapoptotic Bcl-2 family members such as Bax and Bak, which convey death signals (15, 16). Almost two dozen Bcl-2 family proteins have been identified (17). Several pathogens have been shown to manipulate the activity and availability of caspases and Bcl-2 family proteins, thus manipulating cell death pathways.

Although the mechanisms of macrophage apoptosis have been described for a variety of bacteria, including Chlamydia (2), the underlying mechanisms by which GBS induces apoptosis in macrophages have not been investigated. Classical apoptotic regulators and effectors, for example caspase-3 and Bcl-2 family members, have been identified in other models of bacterial-induced host cell apoptosis as critical mediators of this apoptotic response. In this study, we have evaluated the role and regulation of caspases, cytochrome c, Bcl-2 family members, and other apoptotic regulators in the GBS-macrophage interaction. We show that GBS-induced macrophage apoptosis is caspase-3 dependent and that GBS provokes unique changes in the apoptotic regulators Bad, 14-3-3, and Omi/high-temperature requirement A2 (HtrA2).

In this study, we used GBS 874391, a virulent serotype III-3 strain described elsewhere (18). GBS 874391 induces a macrophage response involving DNA fragmentation, positive TUNEL staining, and morphological changes consistent with a delayed host cell apoptotic response (14). GBS 874391 was grown in Todd Hewitt broth. The murine macrophage-like cell line J774A.1 (ATCC TIB-67) was maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented with 25 mM HEPES, 2 mM l-glutamine, 10% heat-inactivated FBS, 0.5 μM 2-ME (tissue culture medium (TCM)), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen Life Technologies). For infection, macrophages were seeded into 24-well plates (Corning Incorporated) at 2.5 × 105 cells/well and incubated at 37°C in 5% CO2 for 24 h. Monolayers were washed twice with PBS to remove antibiotics and loosely adherent cells, and macrophages were inoculated with GBS resuspended in TCM at a multiplicity of infection (MOI) of 100 bacteria per macrophage. For preparation of inocula, GBS was grown in Todd Hewitt broth overnight, back diluted 1/10 and grown to late-log phase (2–3 h), washed three times in PBS (3500 × g, 10 min, 22°C), and resuspended to 3.5 × 107 CFU/ml in TCM. Bacterial numbers were determined by OD at 600 nm (Spectronic Genesys 20; Milton Roy) and confirmed retrospectively in each experiment by colony counts on agar. Infection was performed for 2 h followed by addition of fresh TCM with penicillin, streptomycin, and gentamicin (100 μg/ml) to kill extracellular bacteria (14). To obtain a time course of intracellular GBS survival, monolayers (n = 3) were washed twice with PBS, lysed with 0.01% Triton X-100 in distilled water, diluted in PBS, and dispensed onto Todd Hewitt agar for quantitative colony counts. For lysis, macrophage monolayers were incubated in 0.2 ml of 0.01% Triton X-100 for 5 min, scraped to dissociate macrophages, supplemented with 0.8 ml of PBS, vigorously pipetted, and then further diluted in PBS to avoid potential cytotoxic effects of Triton X-100 on GBS. Additional wells were used to assess macrophage viability by trypan blue dye exclusion and for apoptosis analyses.

Thioglycolate-elicited peritoneal macrophages were harvested from C57BL/6 mice (6–8 wk old) purchased from The Jackson Laboratory. Peritoneal macrophages were collected, cultured, and infected as previously described (13). Ethics approval for animal experimentation was obtained from the St. Jude Institutional Animal Care and Use Committee.

For caspase-inhibition, the caspase-3 specific inhibitor Ac-DEVD.CHO (Calbiochem) was added to macrophages at a final concentration of 350 μM 1 h before infection and maintained for the duration of assay. The cell-permeable formulation of the caspase-3 inhibitor DEVD.CHO (Calbiochem) was also used in some experiments as indicated.

To investigate whether GBS-induced macrophage apoptosis involves activation of the caspases, protease activity assays specific for caspase-3, -8, and -9 were measured in GBS-infected macrophages. Caspase-3, -8, and -9 enzyme activity was determined by substrate cleavage analysis using infected and control macrophage lysates with sequence-specific competitive peptide inhibitors. Reagents and plates used for caspase enzyme assays were purchased from Calbiochem. The following substrates were used: for caspase-3, Ac-DEVD-pNA; for caspase-8, Ac-IETD-pNA; and for caspase-9, LEHD-pNA. Briefly, macrophages were infected with GBS, and whole cell lysates were prepared at specific intervals after infection by collecting cells (1,000 × g, 10 min, 4°C), washing cells in PBS, and resuspending cell pellets in ice-cold lysis buffer (Calbiochem) for 5 min on ice. Lysates were clarified (10,000 × g, 10 min, 4°C), aliquoted, and stored at −80°C until assayed. Total protein concentration in lysates was determined using Ettan reagents from the 2-D Quant kit (Amersham Biosciences) with BSA serving as a standard. For caspase enzyme assays, samples were quickly thawed and equal amounts of protein were mixed with substrate alone or with a combination of substrate and specific inhibitor and incubated at 37°C. Reactions were developed with p-nitroaniline at specific intervals to generate specific activity curves for each sample. The amount of substrate cleavage at each time point was measured by absorbance at 405 nm using a microtiter plate reader (Molecular Devices). Samples derived from 5 μM staurosporine-treated J774A.1 macrophages and samples of recombinant human caspase-3, -8, and -9 were used as controls for the assay. Samples were normalized to total protein content within each sample, and data are presented as specific activity (nanomoles per minute or picomoles per minute), which represents the increase in substrate cleavage over time. Each sample was assayed in duplicate, and experiments were repeated at least three times. Mean SEM values are shown. Caspase-3 activation triggered by GBS was confirmed by immunofluorescence microscopy analysis of infected and noninfected peritoneal macrophages from C57BL/6 mice. Macrophages were grown and infected on coverslip inserts in 24-well cell culture plates as previously described (13) and stained for active caspase-3 using a specific anti-active caspase-3 Ab (1/250) from Promega. Staining was performed according to the manufacturers’ instructions using anti-rabbit IgG secondary Ab (1/500; Jackson ImmunoResearch Laboratories) and TO-PRO-3 (Molecular Probes) costaining for identification of macrophage nuclei. Procedures for immunofluorescence microscopy are described below.

To evaluate whether GBS causes relocalization of mitochondrial cytochrome c to the cytosol, J774A.1 cells were infected and cell fractions (mitochondrial and cytoplasmic) were prepared from infected and uninfected cells at 4, 24, and 48 h according to the protocol for the cytochrome c ELISA kit (5265; MBL). Assays were performed in duplicate on two separately prepared cell fractions, and the data shown are mean values of mitochondrial and cytoplasmic cytochrome c concentrations in GBS-infected, staurosporine-treated, or control macrophages. Relocalization of mitochondrial cytochrome c triggered by GBS was confirmed by immunofluorescence analysis of murine peritoneal macrophages. Macrophages were cultured as above and stained with 4 μM Mitotracker Red 580 (Molecular Probes) for 30 min at 37°C in 5% CO2 to identify mitochondria, as well as with mouse anti-rat cytochrome c mAb (Zymed Laboratories; 1/50) and FITC-conjugated anti-mouse secondary IgG (1/500; Jackson ImmunoResearch Laboratories). Staining was performed according to the manufacturer’s instructions, and immunofluorescence microscopy was performed as described below.

To assess whether GBS induces changes in mitochondrial membrane potential, GBS-infected and uninfected J774 macrophages were prepared and stained at specific intervals with the mitochondrial-specific fluorochrome JC-1 (Molecular Probes), which exhibits potential-dependent accumulation in mitochondria as indicated by a fluorescence shift from green to red. Macrophages were incubated with various concentrations of JC-1 ranging from 0.01 to 200 μg/ml (resuspended in TCM) for 30 min at 37°C in 5% CO2. The optimal concentration was determined as 10 μg/ml, which was used for subsequent experiments. Stained cells were washed extensively with PBS and mounted in 20 μl of 0.5 μM TO-PRO-3 medium with 1 mg/ml p-phenylenediamine dihydrochloride (Sigma-Aldrich). Cell mounts were permanently fixed on glass slides and were stored at −20°C until viewing. Sections were viewed using a Leica Microsystems TCS NTSP spectral confocal microscope with a ×62 or ×100 objective; image analyses were performed with Leica Microsystems confocal software.

To analyze the expression and localization of Bcl-2 family proteins in macrophages following GBS infection, we performed Western blots on whole cell lysates and from subcellular fractions enriched for mitochondrial and cytosolic compartments of GBS-infected and uninfected macrophages. Protein extracts of subcellular fractions were prepared using 5 × 107 cells at 4, 24, and 48 h after infection according to the protocols of the ApoAlert cell fractionation kit (BD Clontech). Proteins were quantitated with Ettan reagents (Amersham Biosciences). Proteins (50 ìg) were separated on 15% Tris-SDS PAGE gels run overnight at 5 V/cm in Tris-SDS-glycine running buffer. Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell Microscience) by use of a semidry transfer apparatus (Bio-Rad) at 20 V for 70 min. Blots were blocked overnight in 5% skim milk in 0.1% PBS-Tween 20 (PBST) and probed with specific Abs diluted in 5% skim milk in PBST. Blots were probed with the following Abs overnight: apoptosis-inducing factor at 1/1000 (AB16501) (Chemicon International); heat shock protein (Hsp) 10 at 1/5000 (611779), Hsp60 at 1/5000 (611563), Bcl-xL at 1/500 (610210), and Bad at 1/500 (610392) (BD Pharmingen); p53 at 1/2000 (PC35) and poly-ADP-ribose polymerase (PARP) at 1/600 (512734) (Calbiochem); Bim at 1/1000 (2065) (ProSci); Omi/HrtA2 at 1/4000 (AF1458) (R&D Systems); 14-3-3 at 1/500 (sc-13959), Bak at 1/200 (sc-7873), and Bax at 1/500 (sc-493) (Santa Cruz Biotechnology). A mAb specific for phosphorylated Bad (Ser112) was used at 1/1000 dilution (7E11) and for cytochrome c at 1/500 (4272) (Cell Signaling Technology). Blots were washed extensively in PBST and incubated with HRP-conjugated anti-rabbit, anti-sheep, or anti-mouse IgG for 1 h as secondary Ab in 5% skim milk in PBST, washed extensively in PBST and developed using the ECL Western blotting analysis system with Hyperfilm MP (Amersham Biosciences). Abs against β-actin (1/10,000, A-5441; Sigma-Aldrich) and cytochrome c oxidase subunit IV (COX4) (1/500, K2016–1; BD Clontech), which is present exclusively in the space between the inner and outer mitochondrial membranes, were used as controls for protein loading and cell fractionation, respectively. Immunoblotting was performed on three separately prepared protein samples from three independent experiments; representative results are shown.

GBS-infected and uninfected J774A.1 macrophages were stained using a triple-stain procedure with Mitotracker Red 580 to identify mitochondria, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) to identify the protein of interest, and TO-PRO-3 to identify nuclei. Macrophages (2.5 × 105 cells/well) were grown in 24-well cell culture plates on 15-mm sterile cell culture Thermanox plastic coverslips (174969; Nalge Nunc International) for 24 h and infected with GBS at an MOI of 100. At 4 and 24 h, cell culture medium was removed and cells were incubated in 4 μM Mitotracker Red 580 in TCM for 30 min at 37°C in 5% CO2. Titration analysis demonstrated that this concentration was optimal for staining of J774A.1 macrophages with minimal background. Cells were then gently washed three times in PBS and fixed in 3.7% paraformaldehyde (Sigma-Aldrich) in TCM for 15 min at 37°C in 5% CO2. Cells were washed three times in PBS and permeabilized with ice-cold acetone for 5 min at room temperature. Nonspecific binding sites were blocked with 2% goat serum in PBS for 1 h at room temperature, followed by incubation with either Bad-specific rabbit polyclonal anti-mouse IgG at 1/80 dilution (Santa Cruz Biotechnology) or 14-3-3-specific rabbit polyclonal anti-mouse IgG at 1/60 dilution (Santa Cruz Biotechnology) in 2% goat serum in PBS for 1 h at room temperature. Cells were washed three times in PBS, and Alexa Fluor 488-conjugated goat anti-rabbit IgG was added at 1/500 dilution for 1 h at room temperature. Cells were then washed three times in PBS and mounted in 20 μl of 0.5 μM TO-PRO-3 medium with 1 mg/ml p-phenylenediamine dihydrochloride (Sigma-Aldrich). Cells were viewed using a Leica Microsystems TCS NTSP spectral confocal microscope with a ×62 or ×100 objective and Leica Microsystems confocal software. Image analysis was performed using Image-Pro Plus software version 4.5.1.29 for Windows 98 (Media Cybernetics). Wavelength emission spectra were quantified by measuring four individual fields in four separate quadrants and averaging these values. Each quadrant contained 40–80 cells. Mean fluorescence intensity was compared for infected and control cells. Data for fluorometric analysis are given as the percent increase in fluorescence per pixel of cellular area in GBS-infected macrophages vs controls.

Macrophage viability was compared using the nonparametric Kruskal-Wallis test (SPSS version 9.0). Caspase activity (fold increase) in infected macrophages was compared with that in uninfected controls using a one-sample t test. Mean fluorescence intensity for wavelength emission spectra from confocal studies was compared using ANOVA. Values of p <0.05 were considered significant.

Analysis of caspase-3, -8, and -9 activity in J774A.1 macrophages infected with GBS 874391 at an MOI of 100 demonstrated that infection causes significant increases in the activity of caspase-3 and caspase-9 following infection (Fig. 1). Compared with (uninfected) control macrophages, the increase in caspase-3 activity of GBS-infected macrophages was 4.4- ± 2.4-fold (p = 0.009) by 24 h after infection (Fig. 1). As expected, experiments performed using caspase-specific inhibitors prevented substrate cleavage, confirming the specificity for the observed changes in caspase specific activity (data not shown). GBS also triggered dose-dependent death and activation of caspase-3 in primary murine peritoneal macrophages, as detected by immunofluorescence microscopy analysis (Fig. 1). Kinetics of murine peritoneal macrophage mortality and intracellular GBS survival and aspects of NO regulation of GBS-induced apoptosis are reported elsewhere (13). Hence, GBS induces significant increases in both caspase-3 and caspase-9 activity in macrophages following infection. By contrast, there were no significant increases in caspase-8 activity detected in macrophages after infection (data not shown).

FIGURE 1.

Caspase-3 and caspase-9 are activated following GBS infection of J774A.1 macrophages. Macrophages were infected with GBS 874391, and lysates were prepared at the indicated intervals following infection according to the protocols described in Materials and Methods. Caspase enzyme activity specific for caspase-3 (A) and caspase-9 (B) were measured by substrate cleavage analysis, with extracts from uninfected macrophages (control), staurosporine-treated macrophages (Stauro; 6–14 h at 5 μM), and recombinant human caspase-3 (Recom; C3) used as controls. Compared with (uninfected) control macrophages, the increase in GBS-infected macrophage lysate-mediated substrate-specific cleavage for caspase-3 was 4.4 ± 2.4-fold (p = 0.009) at 24 h (A). Increases for caspase-3 and -9 at 48 h were 1.7 ± 0.7-fold and 1.9 ± 0.1-fold (p = 0.009), respectively. Specificity for protease activity was confirmed by incubation of samples and substrates with specific inhibitors, which prevented substrate cleavage demonstrating caspase specificity (data not shown). C, Immunofluorescence microscopy analysis confirmed that GBS triggers caspase-3 activation in murine peritoneal macrophages (48 h).

FIGURE 1.

Caspase-3 and caspase-9 are activated following GBS infection of J774A.1 macrophages. Macrophages were infected with GBS 874391, and lysates were prepared at the indicated intervals following infection according to the protocols described in Materials and Methods. Caspase enzyme activity specific for caspase-3 (A) and caspase-9 (B) were measured by substrate cleavage analysis, with extracts from uninfected macrophages (control), staurosporine-treated macrophages (Stauro; 6–14 h at 5 μM), and recombinant human caspase-3 (Recom; C3) used as controls. Compared with (uninfected) control macrophages, the increase in GBS-infected macrophage lysate-mediated substrate-specific cleavage for caspase-3 was 4.4 ± 2.4-fold (p = 0.009) at 24 h (A). Increases for caspase-3 and -9 at 48 h were 1.7 ± 0.7-fold and 1.9 ± 0.1-fold (p = 0.009), respectively. Specificity for protease activity was confirmed by incubation of samples and substrates with specific inhibitors, which prevented substrate cleavage demonstrating caspase specificity (data not shown). C, Immunofluorescence microscopy analysis confirmed that GBS triggers caspase-3 activation in murine peritoneal macrophages (48 h).

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GBS infection induced significant cell death of macrophages, such that by 2 days after infection, 27 ± 5% of these cells had perished (vs 14 ± 2% uninfected controls, p < 0.05; see Fig. 2, inset), consistent with previous findings (14). Because caspase-3 represents a terminal effector caspase, we next determined whether inhibition of caspase-3 might protect J774A.1 macrophages from GBS-induced apoptosis. GBS-infected macrophages incubated with the caspase-3-specific inhibitor DEVD displayed significantly increased survival compared with infected untreated macrophages (16 ± 4% vs 27 ± 5% mortality at 48 h, representing a 41% reduction, p = 0.029; Fig. 2). Prolonged intracellular survival of GBS (Fig. 2, inset) is consistent with previous reports (8, 9). Thus, GBS activates caspase-3 and caspase-9 in macrophages, and caspase-3 activity is essential for GBS-induced apoptosis.

FIGURE 2.

Caspase-3 is essential for GBS-induced macrophage apoptosis. Macrophages were pretreated with 350 μΜ DEVD.CHO for 60 min and then infected with GBS 874391 as described in Materials and Methods. Infected macrophages were incubated in media supplemented with DEVD.CHO, and mortality was assessed by trypan blue dye exclusion. Significantly less macrophage cell death was observed in GBS-infected macrophages treated with DEVD.CHO compared with infected untreated controls (∗, p = 0.029). Data represent mean ± SEM for four independent experiments. Inset, Kinetics of macrophage mortality for both infected and noninfected controls (% trypan blue-positive cells) and intracellular GBS survival (log10 CFU/ml) (mean values ± SD, n = 4).

FIGURE 2.

Caspase-3 is essential for GBS-induced macrophage apoptosis. Macrophages were pretreated with 350 μΜ DEVD.CHO for 60 min and then infected with GBS 874391 as described in Materials and Methods. Infected macrophages were incubated in media supplemented with DEVD.CHO, and mortality was assessed by trypan blue dye exclusion. Significantly less macrophage cell death was observed in GBS-infected macrophages treated with DEVD.CHO compared with infected untreated controls (∗, p = 0.029). Data represent mean ± SEM for four independent experiments. Inset, Kinetics of macrophage mortality for both infected and noninfected controls (% trypan blue-positive cells) and intracellular GBS survival (log10 CFU/ml) (mean values ± SD, n = 4).

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Caspase-9 is activated after the release of cytochrome c from mitochondria (19, 20, 21). To determine whether GBS induced release of cytochrome c from mitochondria, we performed a cytochrome c-specific ELISA using mitochondrial and cytosolic fractions of GBS-infected and uninfected J774A.1 macrophages. Indeed, there were obvious reductions in mitochondrial cytochrome c levels in GBS-infected macrophages compared with control uninfected cells (Fig. 3,A); however, levels of cytochrome c released into the cytoplasm were less than those provoked by treating cells with staurosporine (positive control). However, GBS also caused a marked depolarization of mitochondrial membrane potential in infected macrophages, as indicated by the detection of J-aggregates in control cells but not in infected cells (Fig. 3,B). GBS also triggered relocalization of mitochondrial cytochrome c in murine peritoneal macrophages, as detected by immunofluorescence microscopy analysis (Fig. 3 C; 24-h data shown). Thus, GBS triggers significant alterations in mitochondrial function in macrophages, suggesting involvement of the intrinsic pathway of apoptosis in GBS-triggered cell death.

FIGURE 3.

GBS causes alterations in macrophage mitochondrial functions. Macrophages were infected with GBS 874391; at 48 h, cell fractions (mitochondrial, cytoplasmic) were prepared and applied to a cytochrome c-specific ELISA (A) to measure cytochrome c in each fraction. Experiments were repeated twice, and data given are means ± SEM. Depletion of mitochondrial cytochrome c and increased cytoplasmic cytochrome c were observed in infected macrophages. Staurosporine-treated macrophages showed a more marked response. B, Change in mitochondrial membrane potential following infection was assessed by JC-1 mitochondrial staining (5 μg/ml shown). Under normal (polarized) conditions, JC-1 monomers form aggregates in cells and these emit at the red wavelength. Mitochondrial membrane depolarization causes JC-1 to remain in monomeric form only, which emits at the green wavelength. Potential-dependent accumulation of J-aggregates is shown in normal cells with a fluorescence shift from green to red, which is lost following infection with GBS. C, Immunofluorescence microscopy analysis using colocalization staining with Mitotracker Red 580 confirmed GBS-triggered relocalization of mitochondrial cytochrome c to the cytosol in murine peritoneal macrophages. Ctrl, control.

FIGURE 3.

GBS causes alterations in macrophage mitochondrial functions. Macrophages were infected with GBS 874391; at 48 h, cell fractions (mitochondrial, cytoplasmic) were prepared and applied to a cytochrome c-specific ELISA (A) to measure cytochrome c in each fraction. Experiments were repeated twice, and data given are means ± SEM. Depletion of mitochondrial cytochrome c and increased cytoplasmic cytochrome c were observed in infected macrophages. Staurosporine-treated macrophages showed a more marked response. B, Change in mitochondrial membrane potential following infection was assessed by JC-1 mitochondrial staining (5 μg/ml shown). Under normal (polarized) conditions, JC-1 monomers form aggregates in cells and these emit at the red wavelength. Mitochondrial membrane depolarization causes JC-1 to remain in monomeric form only, which emits at the green wavelength. Potential-dependent accumulation of J-aggregates is shown in normal cells with a fluorescence shift from green to red, which is lost following infection with GBS. C, Immunofluorescence microscopy analysis using colocalization staining with Mitotracker Red 580 confirmed GBS-triggered relocalization of mitochondrial cytochrome c to the cytosol in murine peritoneal macrophages. Ctrl, control.

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Immunoblotting of a range of proteins that regulate apoptosis using protein extracts from whole cells, as well as from subcellular fractions of infected and uninfected macrophages, was performed to gain insight into the spectrum of the apoptotic response triggered by GBS. Analysis of whole cell lysates demonstrated increases in the total amount of the proapoptotic Bad protein in GBS-infected macrophages, which was apparent as early as 4 h following infection (Fig. 4). In contrast, total levels of Bax were not altered in infected macrophages compared with control cells (Fig. 4). GBS also caused obvious decreases in the total amount of PARP in macrophages, although no typical 85-kDa PARP cleavage product was detected at the time points analyzed (but was detected in staurosporine-treated cells) (Fig. 4). GBS did not cause decreases in the total levels of cytochrome c present within macrophages (Fig. 4). Expression of antiapoptotic Bcl-2 family proteins, including Bcl-2, was also not reduced during GBS infection (Fig. 4 and data not shown).

FIGURE 4.

GBS up-regulates total levels of proapoptotic Bad and induces cleavage of PARP, following infection of macrophages. Western blots were prepared using 50 μg of protein from whole cell lysates of GBS-infected and uninfected (control; Con) macrophages. Blots were probed with anti-mouse-specific Abs to determine total levels of Bad, Bax, PARP, Bcl-2, and cytochrome c in macrophages following infection. Actin was used as a loading control. Increased expression of Bad induced by GBS at 4 h is followed by a decrease in total full-length PARP at 48 h. No changes in the steady-state levels of Bax or Bcl-2 were observed after infection. Results shown are representative of two independent experiments.

FIGURE 4.

GBS up-regulates total levels of proapoptotic Bad and induces cleavage of PARP, following infection of macrophages. Western blots were prepared using 50 μg of protein from whole cell lysates of GBS-infected and uninfected (control; Con) macrophages. Blots were probed with anti-mouse-specific Abs to determine total levels of Bad, Bax, PARP, Bcl-2, and cytochrome c in macrophages following infection. Actin was used as a loading control. Increased expression of Bad induced by GBS at 4 h is followed by a decrease in total full-length PARP at 48 h. No changes in the steady-state levels of Bax or Bcl-2 were observed after infection. Results shown are representative of two independent experiments.

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Bad exerts its proapoptotic affects following translocation to the mitochondria (22, 23), and we therefore assessed whether GBS also triggered changes in Bad localization. For these analyses, controls to confirm proper subcellular fractionation (COX4) and protein loading (β-actin) showed successful separation of cytoplasmic and mitochondrial compartments of infected and control macrophages (COX4 in mitochondrial fraction only; Fig. 5). Among the proteins analyzed, the most marked alterations in expression or subcellular localization following GBS infection were for Bad, Bax, 14-3-3, Bcl-xL, and Omi/HtrA2 (Fig. 5). Specifically, GBS infection provoked increases in the amount of mitochondrial-associated Bax and increased levels of cytosolic and mitochondrial Bad. Bad activity is inhibited by phosphorylations that direct its association with the 14-3-3 chaperone protein (24, 25, 26). Assessment of the phosphorylation status of Bad (P-Bad) with a mAb directed against the phospho-Ser112 residue demonstrated decreased levels of phospho-Bad Ser112 following infection (Fig. 5), consistent with the notion that GBS also activates Bad’s apoptotic functions. The amount of 14-3-3 associated with the mitochondrial fraction was also markedly increased following infection, and increased levels of cytosolic Bcl-xL were also induced by GBS. In contrast, levels of Omi/HtrA2, a mitochondrial proapoptotic serine protease that induces both caspase-dependent and caspase-independent cell death (reviewed in Ref. 27) were reduced following infection of macrophages with GBS compared with control cells. Furthermore, Western blots of subcellular fractions confirmed depletion of mitochondrial cytochrome c following infection (see data from Fig. 3), although only a modest increase in cytosolic cytochrome c was detected at 24 h following infection (Fig. 5). No consistent changes in the expression or translocation of other apoptotic regulators were observed following infection, including Bak, Bim, apoptosis-inducing factor, p53, Hsp10, and Hsp60 (Fig. 5).

FIGURE 5.

Expression and subcellular localization of apoptotic regulators in J774A.1 macrophages following GBS infection. Immunoblots of subcellular fractions enriched for mitochondria and cytosol were used to determine protein levels and changes in their localization following infection. Increased expression of Bad in the cytosol of GBS-infected macrophages is consistent with immunoblot analyses. The proapoptotic protein Omi/HtrA2 was down-regulated at 24 h in infected macrophages. Simultaneously, levels of the Bad antagonist 14-3-3 increased in mitochondria fractions, but this is a futile response given dephosphorylation of Bad in GBS-infected cells (shown as a decrease in Ser112 phospho-Bad (P-Bad) in mitochondria at 24 h). Increased localization of Bax was also observed in the mitochondria of infected cells. There were no changes in the expression of several other apoptosis regulator proteins, including Bak, Bim, AIF, p53, Hsp10, and Hsp60.

FIGURE 5.

Expression and subcellular localization of apoptotic regulators in J774A.1 macrophages following GBS infection. Immunoblots of subcellular fractions enriched for mitochondria and cytosol were used to determine protein levels and changes in their localization following infection. Increased expression of Bad in the cytosol of GBS-infected macrophages is consistent with immunoblot analyses. The proapoptotic protein Omi/HtrA2 was down-regulated at 24 h in infected macrophages. Simultaneously, levels of the Bad antagonist 14-3-3 increased in mitochondria fractions, but this is a futile response given dephosphorylation of Bad in GBS-infected cells (shown as a decrease in Ser112 phospho-Bad (P-Bad) in mitochondria at 24 h). Increased localization of Bax was also observed in the mitochondria of infected cells. There were no changes in the expression of several other apoptosis regulator proteins, including Bak, Bim, AIF, p53, Hsp10, and Hsp60.

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To confirm these analyses, we stained GBS-infected macrophages with Bad-specific rabbit anti-mouse IgG and Alexa Fluor 488 anti-rabbit IgG in combination with Mitotracker Red 580 and TO-PRO-3 nuclear staining. Fluorometric analysis of confocal images confirmed that GBS-infected macrophages displayed significant (p = 0.01) increases in the expression of Bad following infection (Fig. 6,A). Mitotracker Red staining of macrophage mitochondria also showed relocalization of Bad to the mitochondria in GBS-infected macrophages (Fig. 6). Interestingly, visualization of Bad in GBS-infected macrophages also suggested some degree of nuclear relocalization during infection, which was not observed in control macrophages (Fig. 6).

FIGURE 6.

Expression and subcellular localization of Bad and 14-3-3 during GBS infection. Macrophages were infected with GBS and stained using Mitotracker Red 580, goat anti-rabbit Alexa Fluor 488 (against protein-specific rabbit polyclonal anti-mouse IgG), and TO-PRO-3 to identify mitochondria (red), Bad (green), and nuclei (blue), respectively. Confocal microscopy analyses demonstrated a significant increase in fluorescence intensity for Bad (A) and 14-3-3 (B) in macrophages infected with GBS compared with control cells (∗, p = 0.01 at 24 h). Some colocalization of Bad with mitochondria was observed in macrophages, confirming immunoblot analyses.

FIGURE 6.

Expression and subcellular localization of Bad and 14-3-3 during GBS infection. Macrophages were infected with GBS and stained using Mitotracker Red 580, goat anti-rabbit Alexa Fluor 488 (against protein-specific rabbit polyclonal anti-mouse IgG), and TO-PRO-3 to identify mitochondria (red), Bad (green), and nuclei (blue), respectively. Confocal microscopy analyses demonstrated a significant increase in fluorescence intensity for Bad (A) and 14-3-3 (B) in macrophages infected with GBS compared with control cells (∗, p = 0.01 at 24 h). Some colocalization of Bad with mitochondria was observed in macrophages, confirming immunoblot analyses.

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The induction of Bcl-xL and 14-3-3 and the reductions in Omi/HtrA2 are most consistent with a response of the macrophage to prevent apoptosis. However, in the face of increases in dephosphorylated Bad and relocalization of Bax to the mitochondria and subsequent release of cytochrome c and activation of caspase-9 and caspase-3, this appears to be a futile response.

Macrophage apoptosis triggered by bacterial infection has been described for a number of pathogens, including Salmonella, Shigella, and Streptococcus (3). The mechanisms of apoptosis have been described for several of these infections, and the ways in which different bacteria manipulate the host cell apoptotic machinery are a subject of intense interest, as understanding the role of apoptosis may lead to new potential therapeutic strategies. Although Streptococcus agalactiae induces apoptosis in macrophages, the molecular mechanisms of GBS-induced macrophage apoptosis are unknown. In this study, we showed that this pathogen activates caspase-9 and caspase-3 and that caspase-3 is an essential mediator of GBS-induced apoptosis. These findings suggest the involvement of the intrinsic mitochondrial pathway of programmed cell death in GBS-mediated apoptosis rather than signaling through death receptors (28, 29, 30). In accord with this notion, we found that this pathogen triggers alterations of mitochondria typical of this response, including release of cytochrome c and alterations in mitochondrial membrane potential. Furthermore, we found that GBS does not activate caspase-8 in J774 macrophages and that CD95 ligation fails to replicate the death response of macrophages triggered by GBS (data not shown), suggesting a limited involvement of extrinsic death receptor signaling pathways. Early activation of caspase-3 at 24 h may reflect procaspase-9 activation at a prior time point that was not tested in this study or, alternatively, may reflect procaspase-9 activation that was not detected at 24 h due to rapid breakdown or conformational modifications resulting from binding to partner proteins such as Apaf-1 (19) so as to impair its detection. Furthermore, it is possible that early activation of caspase-3 by GBS could be due to activation of caspase-10, which can also activate caspase-3 (31).

In a previous study (10), caspase-3 was suggested to be dispensable for GBS-induced apoptosis. However, caspase activity was not directly measured, and the concentrations of the caspase-3 inhibitor Ac-DEVD.CHO that were used (10) were 5-fold lower than those necessary for the cytoprotective effects seen in the current study. Indeed, when GBS-infected macrophages were incubated with Ac-DEVD.CHO at concentrations lower than the 350 μM that was used in this study, no cytoprotective effect was observed. We believe that the high concentrations of Ac-DEVD.CHO necessary to prevent cell death in GBS-infected murine macrophages reflect an inability of the highly hydrophilic chemical inhibitor to readily diffuse across the hydrophobic lipid bilayer of the macrophage membrane. We also assessed the cytoprotective effect of a second caspase-3-specific cell-permeable formulation of DEVD inhibitor in the current study, which was engineered with an additional 16-peptide sequence to increase its hydrophobicity. In these experiments, the cell-permeable caspase-3 inhibitor protects macrophages from GBS-induced apoptosis at lower concentrations than effective doses of Ac-DEVD.CHO. These results, combined with our assays demonstrating that caspase-3 is activated in GBS-infected murine macrophages, establish that caspase-3 contributes to GBS-induced macrophage apoptosis.

A previous study indicated that surface-bound GBS are able to trigger apoptosis in macrophages (10). Whether surface-attached GBS are able to activate caspase-3 and induce the associated changes described in this study is an interesting and unresolved question. It will also be interesting to determine whether there is a correlation between intracellular killing of GBS and macrophage apoptosis, as described for Streptococcus pneumoniae (32). It should also be noted, however, that at present it remains unclear whether the apoptotic outcome of the GBS-macrophage interaction reflects a pathogen-driven virulence mechanism or a host defense mechanism. Future studies are needed to address these aspects of the GBS-macrophage interaction.

In accord with activation of the intrinsic mitochondrial apoptotic pathway, we have also observed changes in the expression and localization of Bcl-2 family members that serve as guardians or executioners of this response. In particular, GBS appears to target the BH3 domain-containing proteins Bax and Bad and leads to increases in Bad expression, to dephosphorylation of Bad, and to relocalization of Bax to mitochondria based on the results of the present study. Typically, monomers of Bax target the mitochondria and form oligomers, which insert deeply into the outer mitochondrial membrane, resulting in membrane permeabilization (33) and leading to cytochrome c release. Such changes in Bax and Bad would effectively compromise the functions of anti-apoptotic proteins such as Bcl-2 or Bcl-xL and would override the futile protective responses engaged by macrophages to spare them from GBS-induced death, which in this study are defined as increases in the expression of Bcl-xL and 14-3-3 and the suppression of Omi/HtrA2.

Omi/HtrA2 normally functions to suppress the inhibitor of apoptosis proteins, which are potent and direct inhibitors of caspase-3. In response to apoptotic stimuli, Omi/HtrA2 translocates from the cytosol to the mitochondria, where it is processed to an active form, and then binds to inhibitor of apoptosis proteins and suppresses their ability to inhibit caspases (34). GBS unexpectedly caused a reduction in Omi/HtrA2 levels, perhaps in another attempt to prevent caspase activation and circumvent host cell death in the face of GBS challenge.

The mechanisms by which GBS provokes increases in the expression of Bad and how these lead to its dephosphorylation are unclear. The effects on Bad expression levels are quite novel, as very few reports suggest changes in the levels of this protein during apoptosis (35, 36); further studies will be required to determine whether the induction of Bad by GBS involves changes in its transcription or posttranscriptional control or even alterations in the half-life of the protein. Bad’s proapoptotic activity is inhibited by its phosphorylation by kinases such as Akt or protein kinase A (37, 38), which then target Bad for interactions with the scaffold protein 14-3-3. The early elevation in Bad is predicted to establish a relative excess of the protein, which cannot, in the long term, be compensated for by increases in the levels of 14-3-3. We believe that the lag time required to establish a relative excess of Bad, in the face of rising 14-3-3 levels, may account for the delayed depletion of cytochrome c from the mitochondria and execution of apoptosis after 48 h. Results of cytochrome c assays demonstrated that mitochondrial cytochrome c release begins to occur in GBS-infected cells as early as 24 h; however, depletion continues over time and is most obvious at later time points (Fig. 3,A). Furthermore, in GBS-infected macrophages, Bad is dephosphorylated, rendering it refractory to the effects of increased levels of 14-3-3, thus allowing Bad free to interact with and neutralize anti-apoptotic proteins, most notably Bcl-2 and Bcl-xL. In turn, this would lead to the activation of Bax, which together with Bak is required for the intrinsic cell death pathway (23, 39), and Bax/Bak activation would then lead to mitochondrial changes that ultimately lead to the activation of caspase-3 and to the death the host macrophage cell (Fig. 7).

FIGURE 7.

Model of GBS-induced macrophage apoptosis. Once GBS gains entry into macrophages (1 ), infection causes a shift in the balance of Bcl-2 family members favoring overexpression of Bad in cytosol and mitochondria, Bax in mitochondria, Bcl-xL in cytosol, and 14-3-3 in mitochondria. There is then a loss of mitochondrial membrane integrity, including membrane depolarization and depletion of cytochrome c from the inner mitochondrial membrane space (2 ). GBS causes activation of caspase-9, and caspase-3 (3 ), the latter of which likely contributes to PARP cleavage (4 ), and is essential for terminal apoptotic processes, including DNA fragmentation (5 ), and cellular disassembly. Depletion of cytosolic Omi/HtrA2 and the induction of Bcl-xL and 14-3-3 are speculated to represent a futile attempt of host cells to survive in the face of GBS challenge. GBS also triggers release of cytokines and NO, the latter of which contributes to the apoptotic response (13 ).

FIGURE 7.

Model of GBS-induced macrophage apoptosis. Once GBS gains entry into macrophages (1 ), infection causes a shift in the balance of Bcl-2 family members favoring overexpression of Bad in cytosol and mitochondria, Bax in mitochondria, Bcl-xL in cytosol, and 14-3-3 in mitochondria. There is then a loss of mitochondrial membrane integrity, including membrane depolarization and depletion of cytochrome c from the inner mitochondrial membrane space (2 ). GBS causes activation of caspase-9, and caspase-3 (3 ), the latter of which likely contributes to PARP cleavage (4 ), and is essential for terminal apoptotic processes, including DNA fragmentation (5 ), and cellular disassembly. Depletion of cytosolic Omi/HtrA2 and the induction of Bcl-xL and 14-3-3 are speculated to represent a futile attempt of host cells to survive in the face of GBS challenge. GBS also triggers release of cytokines and NO, the latter of which contributes to the apoptotic response (13 ).

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We thank K. Gopal Murti and Ken Barnes for technical assistance with confocal microscopy and Klo Spelshouse for digital art.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institute of Allergy and Infectious Diseases Grant RO1 A140918 (to E.E.A.), National Cancer Institute Grant ROI CA76379 (to J.L.C.), the Cancer Center Core Grant 21765, and the American Lebanese Syrian Associated Charities.

3

Abbreviations used in this paper: GBS, Group B Streptococcus agalactiae; PARP, poly-ADP-ribose polymerase; TCM, tissue culture medium; PBST, PBS-Tween 20; Hsp, heat shock protein; COX4, cytochrome c oxidase subunit IV; HtrA2, high-temperature requirement A2; MOI, multiplicity of infection

1
Dockrell, D. H..
2001
. Apoptotic cell death in the pathogenesis of infectious diseases.
J. Infect.
42
:
227
-234.
2
Perfettini, J. L., J. C. Reed, N. Israel, J. C. Martinou, A. Dautry-Varsat, D. M. Ojcius.
2002
. Role of Bcl-2 family members in caspase-independent apoptosis during Chlamydia infection.
Infect. Immun.
70
:
55
-61.
3
Navarre, W. W., A. Zychlinsky.
2000
. Pathogen-induced apoptosis of macrophages: a common end for different pathogenic strategies.
Cell Microbiol.
2
:
265
-273.
4
Gao, L., Y. Abu Kwaik.
2000
. Hijacking of apoptotic pathways by bacterial pathogens.
Microbes Infect.
2
:
1705
-1719.
5
Baker, C. J..
2000
. Group B Streptococcal infections. D. L. Stevens, and E. L. Kaplan, eds.
Streptococcal Infections. Clinical Aspects, Microbiology, and Molecular Pathogenesis
222
-237. Oxford University Press, New York.
6
Antal, J. M., J. V. Cunningham, K. J. Goodrum.
1992
. Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.
Infect. Immun.
60
:
1114
-1121.
7
Noel, G. J., S. L. Katz, P. J. Edelson.
1991
. The role of C3 in mediating binding and ingestion of group B streptococcus serotype III by murine macrophages.
Pediatr. Res.
30
:
118
-123.
8
Valenti-Weigand, P., P. Benkel, M. Rohde, G. S. Chhatwal.
1996
. Entry and intracellular survival of group B streptococci in J774 macrophages.
Infect. Immun.
64
:
2467
-2473.
9
Cornacchione, P., L. Scaringi, K. Fettucciari, E. Rosati, R. Sabatini, G. Orefici, C. von Hunolstein, A. Modesti, A. Modica, F. Minelli, P. Marconi.
1998
. Group B streptococci persist inside macrophages.
Immunology
93
:
86
-95.
10
Fettucciari, K., E. Rosati, L. Scaringi, P. Cornacchione, G. Migliorati, R. Sabatini, I. Fetriconi, R. Rossi, P. Marconi.
2000
. Group B Streptococcus induces apoptosis in macrophages.
J. Immunol.
165
:
3923
-3933.
11
Ring, A., J. S. Braun, J. Pohl, V. Nizet, W. Stremmel, J. L. Shenep.
2002
. Group B streptococcal β-hemolysin induces mortality and liver injury in experimental sepsis.
J. Infect. Dis.
185
:
1745
-1753.
12
Liu, G. Y., K. S. Doran, T. Lawrence, N. Turkson, M. Puliti, L. Tissi, V. Nizet.
2004
. Sword and shield: Linked group B streptococcal β-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense.
Proc. Natl. Acad. Sci. USA
101
:
14491
-14496.
13
Ulett, G. C., E. E. Adderson.
2005
. Nitric oxide is a key determinant of Group B streptococcal-induced murine macrophage apoptosis.
J. Infect. Dis.
191
:
1761
-1770.
14
Ulett, G. C., J. F. Bohnsack, J. Armstrong, E. E. Adderson.
2003
. β-Hemolysin-independent induction of apoptosis of macrophages infected with serotype III group B Streptococcus.
J. Infect. Dis.
188
:
1049
-1053.
15
Bernardi, P., L. Scorrano, R. Colonna, V. Petronilli, F. Di Lisa.
1999
. Mitochondria and cell death: mechanistic aspects and methodological issues.
Eur. J. Biochem.
264
:
687
-701.
16
Reed, J. C..
1997
. Double identity for proteins of the Bcl-2 family.
Nature
387
:
773
-776.
17
Marsden, V. S., A. Strasser.
2003
. Control of apoptosis in the immune system: Bcl-2, BH3-only proteins and more.
Annu. Rev. Immunol.
21
:
71
-105.
18
Takahashi, S., E. E. Adderson, Y. Nagano, N. Nagano, M. R. Briesacher, J. F. Bohnsack.
1998
. Identification of a highly encapsulated, genetically related group of invasive type III group B streptococci.
J. Infect. Dis.
177
:
1116
-1119.
19
Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, X. Wang.
1997
. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
Cell
91
:
479
-489.
20
Green, D. R., J. C. Reed.
1998
. Mitochondria and apoptosis.
Science
281
:
1309
-1312.
21
Shi, Y..
2004
. Caspase activation: revisiting the induced proximity model.
Cell
117
:
855
-858.
22
Green, D. R., G. Kroemer.
2004
. The pathophysiology of mitochondrial cell death.
Science
305
:
626
-629.
23
Orrenius, S..
2004
. Mitochondrial regulation of apoptotic cell death.
Toxicol. Lett.
149
:
19
-23.
24
Zha, J., H. Harada, E. Yang, J. Jockel, S. J. Korsmeyer.
1996
. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-xL.
Cell
87
:
619
-628.
25
Harada, H., B. Becknell, M. Wilm, M. Mann, L. J. Huang, S. S. Taylor, J. D. Scott, S. J. Korsmeyer.
1999
. Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A.
Mol. Cell
3
:
413
-424.
26
Jin, Z., F. Gao, T. Flagg, X. Deng.
2004
. Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis.
J. Biol. Chem.
279
:
23837
-23844.
27
Garrido, C., G. Kroemer.
2004
. Life’s smile, death’s grin: vital functions of apoptosis-executing proteins.
Curr. Opin. Cell Biol.
16
:
639
-646.
28
Ashkenazi, A., V. M. Dixit.
1998
. Death receptors: signaling and modulation.
Science
281
:
1305
-1308.
29
Thornberry, N. A., Y. Lazebnik.
1998
. Caspases: enemies within.
Science
281
:
1312
-1316.
30
Adams, J. M., S. Cory.
1998
. The Bcl-2 protein family: arbiters of cell survival.
Science
281
:
1322
-1326.
31
Chen, M., J. Wang.
2002
. Initiator caspases in apoptosis signaling pathways.
Apoptosis
7
:
313
-319.
32
Dockrell, D. H., M. Lee, D. H. Lynch, R. C. Read.
2001
. Immune-mediated phagocytosis and killing of Streptococcus pneumoniae are associated with direct and bystander macrophage apoptosis.
J. Infect. Dis.
184
:
713
-722.
33
Sharpe, J. C., D. Arnoult, R. J. Youle.
2004
. Control of mitochondrial permeability by Bcl-2 family members.
Biochim. Biophys. Acta
1644
:
107
-113.
34
Suzuki, Y., K. Takahashi-Niki, T. Akagi, T. Hashikawa, R. Takahashi.
2004
. Mitochondrial protease Omi/HtrA2 enhances caspase activation through multiple pathways.
Cell Death Differ.
11
:
208
-216.
35
Puthalakath, H., A. Strasser.
2002
. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins.
Cell Death Differ.
9
:
505
-512.
36
Paquet, C., E. Schmitt, M. Beauchemin, R. Bertrand.
2004
. Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells.
Apoptosis
9
:
815
-831.
37
Datta, S. R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M. E. Greenberg.
1997
. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
Cell
91
:
231
-241.
38
Downward, J..
2004
. PI 3-kinase, Akt and cell survival.
Semin. Cell Dev. Biol.
15
:
177
-182.
39
Scorrano, L., S. A. Oakes, J. T. Opferman, E. H. Cheng, M. D. Sorcinelli, T. Pozzan, S. J. Korsmeyer.
2003
. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis.
Science
300
:
135
-139.