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Group B Streptococcus Induces Macrophage Apoptosis by Calpain Activation¹

Katia Fettucciari^*, Ilaria Fetriconi^*, Roberta Mannucci, Ildo Nicoletti, Andrea Bartoli^*, Stefano Coaccioli and Pierfrancesco Marconi^2,*

^* Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, Institute of Internal Medicine and Oncologic Sciences, and Medical Clinic, S. Maria Hospital, Didactic and Scientific Division of Terni, Perugia University, Perugia, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (M) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine M apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, -spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced M apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing M apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Group B Streptococcus (GBS),³ a pathogen which causes serious neonatal infections (1), has evolved several strategies to overcome host immune defenses and cause diseases (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). It resists phagocytosis by macrophages (M) and polymorphonuclear cells through an antiphagocytic capsule (1, 2, 3). GBS, like intracellular microorganisms, can survive inside respiratory epithelial cells, endothelial cells (4, 5, 6), and M (7, 8) where to survive it inhibits formation of the phagolysosome (7) or impairs protein kinase C signal transduction (8). It can also kill endothelial cells, epithelial cells, and fibroblasts by -hemolysin (9, 10, 11). We have demonstrated that GBS induces a defect in membrane permeability in murine M and human monocytes and then apoptosis (12) triggered from outside the cell and dependent on GBS -hemolysin expression (12). Recently, Ulett et al. (13) demonstrated that the phagocytosed GBS induces apoptosis in J774, a M-like cell line, and this is mediated by a factor coregulated with -hemolysin.

We have shown that GBS-induced M apoptosis is countered by inhibition of extracellular calcium (Ca²⁺) influx but not inhibition of caspases (12). It is worth noting that dying GBS-infected M, despite lack of caspase activation, have several features of caspase-dependent apoptosis, i.e., rounding, shrinking, and DNA strand breaks and fragmentation (12).

Ring et al. (14) and Leib et al. (15) showed that, during experimental sepsis, GBS induces apoptosis in hepatocytes and in neuronal cells, respectively, suggesting that apoptosis may contribute to GBS pathogenesis, so supporting our hypothesis that induction of M apoptosis by GBS could be a strategy to overcome host immune defenses (12).

Numerous pathogens to evade host immune defenses induce apoptosis which can occur through distinct molecular pathways leading to different functional or pathological consequences (16, 17, 18, 19, 20). Apoptosis can eliminate key defense cells necessary to: eradicate the pathogen, inhibit bacterial replication, promote inflammation which aids clearance or prevents further spread of the pathogen within the tissue (16, 17, 20).

Apoptosis is an active process dependent on signaling events in the dying cells (21, 22, 23). There are two apoptosis modes, caspase dependent and independent (21, 22, 23, 24, 25, 26).

Caspase activation is a hallmark of apoptosis and plays a critical role in the initiation and execution of apoptosis (21, 22, 23). Two proapoptotic signal transduction pathways have been described: extrinsic/receptor-linked apoptotic pathway and intrinsic/mitochondria-mediated apoptotic pathway. Both activate downstream effectors caspase-3 and -7 which lead to apoptosis features. One is receptor-mediated caspase-8 activation, the other is cytochrome c (cyt c)-mediated caspase-9 activation (21, 22, 23).

Caspase-independent apoptotic pathways, like caspase-dependent apoptosis, are mediated by proteases and mitochondria (24, 25, 26). Calpains, Ca²⁺-dependent cysteine proteases, are often activated in caspase-independent apoptosis (27, 28, 29, 30, 31, 32, 33, 34, 35). Calpains are cytosolic proteases and two forms, µ- and m-calpain, are ubiquitously expressed and, respectively, regulated by micromolar and millimolar increases in intracellular Ca²⁺ level (36). Calpains and caspases share several death-related substrates including caspases themselves, cytoskeletal proteins, Bax, and Bid (24, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43). Therefore, they can trigger several apoptotic-related events in a caspase-independent manner. Calpains can also serve as upstream triggers of mitochondrial pathways (24, 25). In fact, they can cleave and activate Bax and Bid (34, 40, 41, 42, 43). These latter then induce the mitochondrial apoptogenic protein release which may lead to activation of caspase-dependent (cyt c, Smac/Diablo) or caspase-independent (apoptosis-inducing factor (AIF), endonuclease G (Endo G)) death effectors (21, 22, 23, 24, 25, 26, 44, 45, 46, 47, 48, 49).

Because bacterial apoptosis mechanisms can influence pathogenesis and disease progression (17, 18, 19, 20), the knowledge of cell death pathways induced by pathogens in immune cells, and their regulation, is critical for insight into the role of apoptosis in infections and development of new therapeutic strategies. Because the apoptotic signaling induced by GBS is still unclear, the aim of this study was to characterize mechanisms that underlie GBS-induced M apoptosis. To this end, the role of Ca²⁺-sensitive calpains and the downstream events were analyzed. Our results indicate that, during M apoptosis induction, GBS causes a massive intracellular Ca²⁺ increase which activates the calpains, particularly m-calpain, and this activation is followed by Bax and Bid cleavage and onset of a caspase-independent mitochondrial pathway.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Reagents

The selective inhibitor for µ- and m-calpain directed to the Ca²⁺-binding sites 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606), the 20-fold selective inhibitor for µ-calpain directed to the Ca²⁺-binding sites 3-(5-fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (PD151746), the negative control for calpain inhibitors 2-mercapto-3-phenylpropanonic acid (PD145305), the 27-residue calpastatin peptide (CS peptide, a potent inhibitor of µ- and m-calpain directed to the active site, homologous to the inhibitory domain of calpastatin an endogenous inhibitor of calpains), the leupeptin (inhibitor of µ- and m-calpain directed to the active site), the carbobenzoxy-valyl-phenylalanial (MDL28170, a potent, cell-permeable inhibitor of µ- and m-calpain directed to the active site), the N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN, inhibitor of µ- and m-calpain directed to the active site), the cathepsin B inhibitor IV (CA-074 Me), the ammonium chloride (NH₄Cl, an inhibitor of all lysosomal proteases), the Boc-Asp(OMe)-fluoromethylketone (BAF, a broad-spectrum caspase inhibitor), the clasto-lactacystin -Lactone (clasto-lactacystin, a proteasome inhibitor), the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A (CsA), the staurosporine (STS), and the caspase-3 cellular activity assay kit were obtained from Calbiochem. EGTA, fura 2-AM, propidium iodide (PI), Triton X-100, rabbit polyclonal Ab anti-m-calpain raised against domain I, rabbit polyclonal Ab anti-µ-calpain raised against domain I, and mouse anti--actin mAb were obtained from Sigma-Aldrich. Mouse anti--spectrin (nonerythroid) mAb was purchased from Chemicon International. Rabbit polyclonal Ab anti-Bax (N-20) raised against aa 11–30, mouse mAb anti-Bax (B-9) raised against aa 1–117, goat anti-AIF polyclonal Ab, and rabbit anti-Bcl-x_L polyclonal Ab were obtained from Santa Cruz Biotechnology. Rabbit polyclonal Abs against Bid, caspase-3, and caspase-7 were obtained from Cell Signaling Technology. Rat anti-APAF-1 mAb and rabbit anti-Endo G polyclonal Ab were purchased from Alexis Biochemicals. Mouse anti-cyt c mAb was obtained from BD Pharmingen. Mouse anti-Bcl-2 mAb was purchased from DAKO. Mouse anti-cytochrome c oxidase subunit IV (Cox IV) mAb was purchased from Molecular Probes. Mouse anti-ubiquitinylated proteins mAb was obtained from Biomol International. HRP-conjugated secondary anti-mouse, anti-rabbit, anti-goat, and anti-rat Ig Abs and the ECL system were purchased from Amersham.

The small-interfering-RNA (siRNA) duplexes specific for: the catalytic subunits of mouse µ-calpain (catalog no. M-0622006-00) and mouse m-calpain (catalog no. M-043027-00), the mouse Bax (catalog no. M-061976-00), the mouse Bid (catalog no. M-058612-00), and the control nontargeting siRNA (catalog no. D-001206-13-05) were obtained from Dharmacon RNA Technologies. The siRNA for each calpain, Bax, and Bid contained four RNA sequences in a pool SmartPool selected from the National Center for Biotechnology Information (NCBI) RefSeq Database by a proprietary algorithm. The control nontargeting pool is a pool of four functional nontargeting siRNAs with guanine cytosine content comparable to that of the functional siRNA but lacking specificity for known gene targets.

Microorganisms

GBS, type III, strain COH31 r/s, clinically isolated from a diabetic septic foot ulcer of an adult, rendered resistant to rifampicin and streptomycin, was provided by Dr. M. Wessel (Channing Laboratory, Boston, MA). GBS was grown in Todd-Hewitt broth (THB; Unipath) at 37°C and aliquots were stored at –70°C until used.

For assays, the GBS was grown in THB overnight and then washed and adjusted photometrically (600 nm) to the desired number of CFU per milliliter. Concentration and purity of inoculum was confirmed by quantitative culture on Islam agar (Unipath) plates containing 5% heat-inactivated horse serum.

For some experiments, GBS adjusted photometrically (600 nm) to the desired number of CFU per milliliter was heat inactivated (0.5 h 80°C; hi-GBS), then extensively washed in PBS. Sterility was confirmed by culture on Islam agar plates.

For some experiments, GBS was grown for 18 h in THB in the presence of 10 mg/ml glucose (gGBS), conditions not allowing hemolytic activity expression (12), and then treated as described above.

Preparation of peritoneal M

Outbred CD-1 mice of both sexes, 8–10 wk old, were obtained from Charles River Breeding Laboratories.

Murine peritoneal M were elicited by i.p. injection of 1 ml of a 10% solution of thioglycolate broth (Difco Laboratories) and cells were recovered 4 days later as previously described (12). Cells were resuspended in cold antibiotic-free RPMI 1640 medium with 5% FCS (complete medium) and cell viability was evaluated by Trypan blue exclusion method.

The purity of thioglycolate-elicited M obtained by washing the peritoneal cavity was >80%, as determined by binding of mAb to CD14 (50) and by nonspecific esterase staining.

Furthermore, when the M were allowed to adhere for 1 h in 6-well tissue-culture plates and the nonadherent cells were removed by washing, the resulting M population was 98% pure as determined by nonspecific esterase staining and by binding of mAb to CD14 (50).

Infection procedure

The infection was performed as previously described (12). We used M (1 x 10⁶/ml) infected with GBS, at a cell:microorganism ratio of 1:100, for 1, 1.5, 2 h, and M infected for 2 h washed and reincubated for 12 and 24 h in complete medium containing 100 U/ml penicillin and 100 µg/ml gentamicin. Control M were incubated in medium for the same times.

Infection of M with hi-GBS or gGBS was as described above.

For experiments with PD150606, PD151746, or PD145305, inhibitors were added at concentrations of 50, 25, and 10 µM to M 1.5 h before GBS infection and kept during the course of experiments at the same concentrations.

For experiments with calpain or other protease inhibitors, CS peptide (50 µM), leupeptin (100 µM), MDL28170 (10 µM), ALLN (10 µM), CA-074 Me (10 µM), NH₄Cl (10 mM), BAF (25 µM), or clasto-lactacystin (2 µM) was added to M 1.5 h before GBS infection and kept during the course of experiments.

For EGTA experiments, 1 mM was added to M during the 2 h infection and EGTA 0.5 mM was kept during the course of experiments.

For CsA experiments, 10 µM was added to M 0.5 h before infection and kept during the course of experiments.

M treated with inhibitors but not infected were controls.

Doses were chosen taking account of inhibitor specificity and cytotoxicity. In fact, M treated with PD150606 (50 µM), PD151746 (50 µM), CS peptide (50 µM), leupeptin (100 µM), MDL28170 (10 µM), ALLN (10 µM), CA-074 Me (10 µM), NH₄Cl (10 mM), BAF (25 µM), or clasto-lactacystin (2 µM) remained as viable as control M (97 ± 2% viability inhibitor-treated M; 98 ± 1.5% untreated M) at all times examined, as evaluated by the trypan blue exclusion method.

In preliminary experiments, we used all the above-reported calpain inhibitors, while in the following experiments, PD150606 was chosen taking account of its high selectivity for calpains over other proteases.

PI uptake assay

At different time points, infected and control M were washed, adjusted to 1 x 10⁶/ml in PBS containing PI (5 µg/ml), incubated at 23°C for 5 min, and analyzed on a FACScan flow cytometer (BD Biosciences).

Apoptosis evaluation

At different times, infected and control M were recovered to detect apoptosis (12). The cell pellets were resuspended in 1 ml of hypotonic fluorochrome solution (50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100). Samples were placed overnight in the dark at 4°C, and the PI fluorescence of individual nuclei measured using a FACScan flow cytometer. Data were processed by a Hewlett-Packard computer and analyzed with lysis software.

Assay for GBS intracellular survival in M

The M pretreated or not with calpain inhibitors were infected for 2 h with GBS as described above in the presence or absence of the calpain inhibitors. After this time, the culture supernatants of infected M were removed by aspiration. M monolayers were washed three times with antibiotic-free medium. To quantify the number of intracellular and adherent GBS, the cells were washed and then lysed with Triton X-100 at a final concentration of 0.1% (v/v) in sterile distilled water. Serial dilutions of lysate from each well were prepared, and 0.1 ml of each dilution was plated on Islam agar. The number of CFU was determined after 24 h incubation under anaerobic conditions.

To kill extracellular bacteria, the cultures of M infected for 2 h were incubated for a further 2 h (time 4 h) in complete medium containing 100 U/ml penicillin and 100 µg/ml gentamicin. Then at 4 h, the supernatants containing antibiotics were removed, cells were washed, and the number of intracellular GBS was quantified as described above and in our previous report (8).

Exposure of GBS to 100 U/ml penicillin and 100 µg/ml gentamicin for 2 h was sufficient to kill 100% of microorganisms.

Intracellular Ca²⁺ levels

M were allowed to adhere on 20-mm coverslips for 1 h. For intracellular Ca²⁺ concentration ([Ca²⁺]_i) analysis, cells were washed and loaded with 1 µM fura 2-AM, 0.025% pluronic, and 250 mM sulfinpyrazone (all from Sigma-Aldrich) in HEPES medium with 2% BSA for 0.5 h at 37°C. Cells were washed twice with HEPES, and the coverslips were mounted on an Attofluor perfusion chamber (Molecular Probes) for microscopic analysis. Infection of M with GBS and hi-GBS was performed as described above directly in the perfusion chamber. Fluorescence images were grabbed through an intensified CCD camera (2400-87 model; Hamamatsu Photonics) and analyzed with a dedicated Image-Analysis system (Mips-Ca; Graftek Italia) on a PowerMac (Apple) personal computer. Interference filters on a computer-driven rotational filter wheel selected alternate excitation wavelengths of 340 and 380 nm from a 100 W Hg fluorescence lamp on an IMT-2 Olympus inverted microscope. Background subtraction was conducted independently at each wavelength, and the ratios (340/380 nm), which are proportional to [Ca²⁺]_i, were calculated for each image and represented as pseudocolor. Mathematical analysis was performed on eight cells per field, selected as interest regions. Mean fluorescence of these interest regions was calculated by the software and values exported and plotted against the time of experiments with the SigmaPlot software. The area under the curve between 1 and 2 h was calculated with the GraphPad/Prism 4.0 package. Friedman’s ANOVA with Dunn’s correction was used for statistical analysis.

Total cell lysis and subcellular fractionation

At different times, infected and control M (10 x 10⁶ cells/sample) were lysed in lysis buffer (10 mM HEPES (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 20 mM -glycerophosphate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium orthovanadate, 1 µg/ml pepstatin A, 10 mM benzamidine, and 10 mM sodium fluoride; Sigma-Aldrich) for 0.5 h at 4°C. Insoluble material was removed by centrifugation at 16,000 x g for 10 min at 4°C.

For subcellular fractionation, infected and control M (10 x 10⁶ cells/sample) were resuspended in Mitobuffer (20 mM HEPES, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 20 mM -glycerophosphate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium orthovanadate, 1 µg/ml pepstatin A, 10 mM benzamidine, and 10 mM sodium fluoride; Sigma-Aldrich). After incubation on ice for 0.5 h, cells were disrupted at 4°C by 20 passages through a 25-gauge needle fitted in a 1-ml syringe. Unbroken cells and nuclei were removed by centrifugation at 700 x g for 5 min at 4°C. The resulting supernatant was further centrifuged at 13,000 x g for 20 min at 4°C, followed by collection of the supernatant (cytosolic fraction) and the pellet (mitochondrial fraction).

Protein content was determined by a standard Bradford protein assay (Bio-Rad).

Immunoblotting

Proteins, 30 µg, were boiled, separated on 10, 12, or 15% SDS-PAGE, and electrophoretically transferred to a nitrocellulose membrane. The blots were blocked with 5% milk in TBST for 1 h, and then incubated overnight at 4°C with an appropriate dilution of the primary specific Abs. The blots were washed four times with TBST and incubated for 1 h with appropriate HRP-conjugated secondary Ab. Immunoreactive bands were developed using ECL. Autoradiography was performed for variable times. As loading controls, the blots were stripped in stripping buffer (100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 6.8)) for 0.5 h at 50°C, and after washing with TBST, reprobed, as described above, with anti--actin mAb or anti-Cox IV mAb followed by incubation with HRP-conjugated Ab. Proteins were detected by ECL.

Measurement of calpain activity by a fluorogenic-substrate assay in intact cells and cell extracts

A fluorogenic-substrate assay, using the calpain-specific and membrane-permeable fluorogenic substrate Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC; Sigma-Aldrich) (51), was used to detect calpain activity in intact cells (30, 34, 51, 52) or cell extracts (53). Proteolytic hydrolysis of the peptidyl-7-amino bond liberates the highly fluorescent AMC moiety. Fluorescence was quantified with a Titertek Fluoroskan II platereader fluorometer with 355 nm excitation and 460 nm emission filters (Flow Laboratories). Standard curves were generated with free AMC (Sigma-Aldrich) for each experiment.

To measure calpain activity in intact cells, M plated in 96-well black plates (Costar) at 10⁵ cells/well in 100 µl of assay buffer (115 mM NaCl, 1 mM KH₂PO₄, 5 mM KCl, 1 mM CaCl₂, 1.2 mM MgSO₄, 25 mM HEPES (pH 7.4)) were preincubated with or without calpain and protease inhibitors then 80 µM Suc-LLVY-AMC was added. Following incubation for 30 min at 37°C 5% CO₂, cells were infected with GBS and the plates were incubated at 37°C in the fluorometer. Fluorescence was measured at 5-min intervals starting immediately after addition of GBS for up to 2 h. Calpain activity was calculated and gave a fold increase over basal activity.

Detection of calpain activity in cell extracts was performed using a calpain activity assay kit (Calbiochem) according to manufacturer’s protocol. This kit contains Suc-LLVY-AMC and optimized extraction and reaction buffers. Briefly, at different times, infected and control M (10 x 10⁶ cells/sample) were lysed in extraction buffer for 0.5 h at 4°C. Clarified cell lysates were then incubated in the presence or absence of calpain inhibitors with the substrate and reaction buffer for 15 min at room temperature and then fluorescence emission was measured. Calpain activity, calculated as nanomoles of AMC released per minute per milligram of protein, was shown as fold increase over basal activity.

Estimation of µ- and m-calpain activity

Casein zymography was applied to detect and resolve µ- and m-calpain activation as described by Raser et al. (54).

At 2 h, infected and control M were suspended at 8 x 10⁷/ml in Ca²⁺- and Mg²⁺-free PBS with protease inhibitors (10 µg/ml aprotinin, 5 µg/ml leupeptin, 1.8 mg/ml iodoacetamide, and 1 µM PMSF; Sigma-Aldrich). Cell lysis was performed by five cycles of liquid nitrogen (LN₂) flash-freezing, followed by rapid thawing of the sample in a 37°C water bath (55). Cell lysates were clarified by short spin at 10,000 x g, flash-frozen in LN₂ and stored at –70°C. Electrophoretic separation of the enzyme from calpastatins which influence its activity was done in a nonreducing, detergent-free buffer in a 12% polyacrylamide gel containing 0.2% milk casein (Sigma-Aldrich) at 126 V (54, 55). The amount of lysate corresponding to 2 x 10⁶ cells was loaded on each lane. The calpain activity in the gel was induced by overnight incubation in a buffer containing 2 mM CaCl₂ and 10 mM DTT in 20 mM Tris-HCl (pH 7.4), at room temperature. Casein proteolysis by calpains produced clear, transparent bands in the Coomassie blue-stained gels. The identity of the bands with the µ- and m-calpain was confirmed by simultaneous running of a sample containing a mixture of purified calpains (0.1 µg of human µ-calpain and 0.1 µg of porcine kidney m-calpain; Calbiochem) in the Ca²⁺- and Mg²⁺-free PBS. Casein-digesting activity was only seen at the levels corresponding to the band positions of these "standard" µ- and m-calpain.

Specificity of the test was confirmed by the fact that no proteolytic activity was seen in the gels incubated in activation buffer without Ca²⁺.

Caspase assay

Detection of caspase-3 and caspase-3-like activity in cell extracts was performed using a caspase-3 cellular activity assay kit (Calbiochem), based on fluorogenic substrate cleavage analysis, according to manufacturer’s protocol. This kit contains Ac-DEVD-AMC, a substrate for caspase-3 and -7 (32, 39), AMC standard, caspase-3 inhibitor I (Ac-DEVD-CHO), human caspase-3 recombinant and optimized lysis and assay buffers. Briefly, at different times, infected and control M (10 x 10⁶cells/sample) were lysed in cold cell lysis buffer for 5 min on ice. Clarified cell lysates were then incubated at 37°C in the presence or absence of caspase inhibitors for 0.5 h in assay buffer. The reaction then was started by adding 50 µM substrate and fluorescence emission was measured at 37°C with a Titertek Fluoroskan II platereader fluorometer with 355 nm excitation and 460 nm emission filters. Data were recorded at 15 min intervals for 2 h. Samples from STS-treated M (5 µM for 14 h) (13, 56), STS- and 25 µM BAF-treated M and samples of human recombinant caspase-3 were used as controls. Caspase-3-like activity was calculated as picomoles of AMC released per minute per microgram of protein. Standard curves were generated with free AMC for each experiment. Protein content of lysates was determined by a standard Bradford protein assay.

siRNA transfection

siRNA transfection was performed according to the manufacturer’s protocol for Lipofectamine 2000 (Invitrogen Life Technologies). Briefly, siRNAs specific for the catalytic subunits of mouse µ- or m-calpain, for the mouse Bax, the mouse Bid, and the control nontargeting siRNA at the concentrations indicated in the text were solubilized and formed complexes separately with a lipid-based transfectant, Lipofectamine 2000. The siRNA-lipofectamine complexes were transfected into the cultured M seeded 18 h before in a 6-well plate at 70–80% cell confluence and incubated for the time indicated in the text. In mock transfection, all vehicles were used except for the siRNA. Throughout the experiments, cell vitality was monitored continuously. The cells were vital throughout the course of all experiments as determined by trypan blue exclusion assay.

At 24, 48, and 72 h posttransfection in preliminary experiments and at 72 h posttransfection in the subsequent experiments, the effect of siRNA was evaluated both by: 1) measuring the relative expression of m-calpain, µ-calpain, Bax, and Bid gene vs GAPDH by quantitative RT-PCR (qRT-PCR); and 2) analyzing the specific protein knockdown by immunoblotting using specific Abs, and -actin as loading control. The density of the bands was quantified, after scanning with Versadoc 1000 (Bio-Rad), by Quantityone software.

Furthermore, at 72 h posttransfection, the cells were washed, placed in fresh RPMI 1640, and infected with GBS as described above and then apoptosis and immunoblot analysis were performed as described above.

qRT-PCR

We used qRT-PCR (57) to quantify the expression of mouse genes by using the following sense and antisense primers: µ-calpain, 5'-gagctttcaggatggctacg-3' and 5'-gcataggctttctccagcag-3'; m-calpain, 5'-ccaagctgttgatgacgaga-3' and 5'-tgaagccgtctgacttgatg-3'; Bax, 5'-catgggctggacactggact-3' and 5'-catgtgggggtcccgaagta-3'; Bid, 5'-catccacaacattgccagac-3' and 5'-tctccatgtctctggggaag-3'; GAPDH, 5'-ctgagtatgtcgtggagtctac-3' and 5'-gttggtggtgcaggatgcattg-3'. All PCR primers were designed using PRIMER3-OUTPUT software using published sequence data from the NCBI database. Total RNA was isolated (TRIzol reagent; Invitrogen Life Technologies) from M at the posttransfection time indicated in the text. Purified RNA (1 µg) was treated with DNaseI (Invitrogen Life Technologies) and then the RNA was reverse transcribed with Superscript II (Invitrogen Life Technologies) in a 20-µl reaction volume using random primers. For qRT-PCR, a 25-ng template was dissolved in a 25-µl mix containing 0.2 µmol/L of each primer, 12.5 µl of platinum 2x SYBR Green qPCR SuperMix-UDG and 0.5 µl of ROX Reference Dye (Invitrogen Life Technologies). All reactions were performed in triplicate. The thermal cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min in the GeneAmp 5700 Sequence Detection System (Applied Biosystems). The fluorescence emission data for each sample were processed using GeneAmp 5700 SDS Software (Applied Biosystems). The mean value of the replicates for each sample was calculated and expressed as the cycle threshold (Ct; cycle number at which each PCR reaches a predetermined fluorescence threshold, set within the linear range of all reactions).

The amount of gene expression was then calculated as the difference (Ct) between the mean Ct value of the sample for the target gene and the mean Ct value of that sample for the housekeeping gene, GAPDH. The relative expression was calculated as the difference (Ct) between the Ct value of the transfected cells vs Ct value of nontransfected cells. The relative expression level was expressed as 2^–Ct (ABI Prism 7700 Sequence Detection System; "Relative Quantification of Gene Expression", Livak Applied Biosystems User Bullettin no. 2 (part number 4303859), 1997).

Statistical analysis

All experiments of PI uptake, apoptosis, and calpain activity assays in the absence or presence of inhibitors were repeated six times. Experiments of silencing by siRNA and caspase assay were repeated four times. Data are presented as the means ± SD of six or four independent experiments performed in triplicate. The data for each experiment were analyzed by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
GBS causes a rise in cytosolic Ca²⁺ during M apoptosis induction

We have shown that GBS first induces alterations in membrane permeability and then M apoptosis (Ref. 12 , and Fig. 1A) and suggested that influx of extracellular Ca²⁺ is part of a critical signaling pathway for GBS-induced M apoptosis, because 70% inhibition of apoptosis was observed when the extracellular Ca²⁺ was chelated by EGTA (Ref. 12 , and Fig. 1B). Therefore, in this study we first examined whether [Ca²⁺]_i increased and the outcome during GBS-induced M apoptosis using the intracellular Ca²⁺ indicator dye, fura 2-AM.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. GBS causes a [Ca2+]i increase during M apoptosis induction. A, Percentage of PI+ cells (lines) by PI uptake assay or apoptosis (histograms) by evaluating percentage of hypodiploid nuclei at flow cytometry was determined in GBS or hi-GBS infected M and controls recovered at different times after infection. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M vs control M) according to Student’s t test. B, Apoptosis in GBS-infected M in the absence or presence of 1 mM EGTA, kept at 0.5 mM during experiments, was measured at 24 h evaluating percentage of hypodiploid nuclei at flow cytometry. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M treated with EGTA vs untreated GBS-infected M) according to Student’s t test. C, Analysis of [Ca2+]i. Images collected at basal state and 2 h of infection were from control, GBS-infected, and hi-GBS-infected M. The [Ca2+]i level is reported as pseudocolor images of 340/380 ratio (scale on right). Blue is the lowest [Ca2+]i and red the highest [Ca2+]i. D, Time-course analysis of [Ca2+]i measured at single-cell levels in control, GBS-infected, and hi-GBS-infected M, in representative experiments.

The difference in [Ca²⁺]_i, expressed as area under the curve of mean fluorescence, was highly significant (p < 0.001) between GBS-treated M (442.0 ± 52.0, mean ± SEM), control cells (79.5 ± 15.0), and hi-GBS-treated M (79.2 ± 20.2).

Calpains are activated and involved in GBS-induced M apoptosis

A possible direct target, by which elevated intracellular Ca²⁺ triggers apoptosis, are calpains, Ca²⁺-dependent proteases, which mediate apoptosis even in the absence of caspase activation (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 58).

To investigate the possible involvement of calpains in GBS-induced M apoptosis, the effect of PD150606, a highly specific inhibitor of calpains (29, 30), was tested. PD150606 inhibited GBS-induced apoptosis in a dose-dependent manner (Fig. 2A). PD150606 at 50 and 25 µM inhibited apoptosis by 80 and 35%, respectively. Instead, 10 µM PD150606 had no significant effect. The PD145305, a negative control for calpain inhibitor (30), did not affect GBS-induced apoptosis (Fig. 2A), so indicating that the effect of PD150606 is due to blocking of the calpain Ca²⁺-binding site.

View larger version (60K): [in this window] [in a new window]   FIGURE 2. Inhibition of GBS-induced M apoptosis and -spectrin cleavage by calpain inhibitor PD150606 and EGTA. A, Effect of PD150606 and PD145305, inactive compound, on apoptosis. M pretreated for 1.5 h in absence or presence of the indicated concentrations of inhibitors and infected with GBS for 2 h were recovered at 24 h, and apoptosis was measured by evaluating the percentage of hypodiploid nuclei at flow cytometry. Treating control M with each concentration of PD150606 or PD145305 for the same time did not induce apoptosis. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M treated with inhibitor vs untreated GBS-infected M) according to Student’s t test. B, Effect of PD151746 on apoptosis. M pretreated for 1.5 h in absence or presence of the indicated concentrations of inhibitor and infected with GBS for 2 h were recovered at 24 h, and apoptosis was measured by evaluating the percentage of hypodiploid nuclei at flow cytometry. Treating control M with each concentration of PD151746 for the same time did not induce apoptosis. Data are means ± SD of six experiments done in triplicate. C, Time course of -spectrin cleavage. Whole-cell lysates from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE and probed with anti--spectrin mAb. -actin mAb was loading control. D, Effect of PD150606 and EGTA on -spectrin cleavage. Whole-cell lysates from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE, and probed with anti--spectrin mAb. -Actin was loading control. Blots in C and D are representative of five independent experiments. Intact protein (solid arrow) and major BP (open arrow) are indicated.

Cleavage of the cytoskeletal protein -spectrin into 150- and 145-kDa breakdown products (BPs) is a characteristic sign of calpain activity (39). Therefore, we analyzed calpain activation during GBS-induced M apoptosis by monitoring the appearance of calpain-specific -spectrin BPs. Immunoblot analysis indicated that GBS activates calpains during M apoptosis induction (Fig. 2C). The calpain-specific 145-kDa BP appeared strongly at 2 h, remained high up to 12 h and disappeared at 24 h. Concomitantly, full-length (f.l.) -spectrin decreased strongly. No caspase-specific 120-kDa -spectrin BP was found at all times examined (Fig. 2C).

Cleavage of -spectrin by calpains was confirmed by experiments with calpain inhibitor PD150606. Pretreatment of M with 50 µM PD150606 before GBS infection prevented already at 2 h both the formation of 145-kDa BP and the decrease of f.l. -spectrin (Fig. 2D). No caspase-dependent 120-kDa BP appeared in PD150606 treated M during GBS-induced apoptosis at 2 h (Fig. 2D), 12 and 24 h (data not shown).

No significant inhibition of -spectrin cleavage was observed with 50 µM PD151746 (data not shown).

Because changes in Ca²⁺ homeostasis are crucial for calpain activity, we used EGTA to determine whether removal of extracellular Ca²⁺ affected the appearance of calpain-specific -spectrin BP in GBS-induced M apoptosis. EGTA prevented the formation of 145-kDa BP and the reduction of f.l. -spectrin (Fig. 2D), demonstrating that calpains in the presence of EGTA are not activated.

To provide further evidence that calpains are responsible for GBS-induced M apoptosis and -spectrin cleavage we also used: 1) four structurally different active site targeting peptidic calpain inhibitors derived from natural sources (CS peptide, leupeptin, MDL28170, ALLN) (36, 59); 2) the cathepsin B inhibitor CA-074 Me; 3) the inhibitor of all lysosomal cathepsins NH₄Cl; 4) the caspase inhibitor BAF; and 5) the proteasome inhibitor clasto-lactacystin.

CS peptide (50 µM), leupeptin (100 µM), MDL28170 (10 µM), and ALLN (10 µM) were all partially inhibitory. In fact, we observed 70% inhibition of GBS-induced M apoptosis (Fig. 3A) and already at 2 h, prevention of -spectrin cleavage into the 145-kDa BP (Fig. 3B). On the contrary, no inhibition of apoptosis (Fig. 3A) or -spectrin cleavage (data not shown) was observed with CA-074 Me (10 µM), NH₄Cl (10 mM), BAF (25 µM), and clasto-lactacystin (2 µM).

View larger version (52K): [in this window] [in a new window]   FIGURE 3. Inhibition of GBS-induced M apoptosis and -spectrin cleavage by active site targeting calpain inhibitors. A, Effect of different calpain inhibitors and a panel of inhibitors of other proteases on apoptosis. M pretreated for 1.5 h in absence or presence of the indicated concentrations of inhibitors and infected with GBS for 2 h were recovered at 24 h and apoptosis measured evaluating the percentage of hypodiploid nuclei at flow cytometry. Treating control M with each inhibitor for the same time did not induce apoptosis. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M treated with inhibitor vs untreated GBS-infected M) according to Student’s t test. B, Effect of active site targeting calpain inhibitors on -spectrin cleavage. Whole-cell lysates from M untreated, pretreated with CS peptide (50 µM), leupeptin (100 µM), MDL28170 (10 µM), ALLN (10 µM), infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE, and probed with anti--spectrin mAb. -Actin was loading control. Blot is representative of three independent experiments. Intact protein (solid arrow) and major BP (open arrow) are indicated.

As positive control to show that 2 µM clasto-lactacystin inhibited proteasome activity in peritoneal murine M, we investigated proteasome inhibition by immunoblotting, analyzing the accumulation of multiubiquitinylated proteins (61). We found an accumulation of high m.w. protein-ubiquitin conjugates in clasto-lactacystin-treated M (data not shown).

To rule out that the calpain inhibitors used impaired the GBS adherence and uptake by M and therefore that the inhibition of apoptosis was due to interference by inhibitors, the number of viable GBS in M was quantified both after 2 h infection and at 4 h (2 h infection plus 2 h incubation with antibiotics). At 2 h, the number of viable GBS was the same in both inhibitor-treated and untreated M (Table I), while at 4 h it was higher in inhibitor-treated M than in untreated M (Table I), likely due to the inhibition of M apoptosis by calpain inhibitors. There was also no direct effect of inhibitors on the GBS, because adding each inhibitor (at the concentration used) to GBS cultures did not affect the viability and growth of GBS which was similar with all inhibitors used (data not shown). These data suggest that the inhibition of M apoptosis by calpain inhibitors was not due to an effect of the inhibitors on GBS infection.

View this table: [in this window] [in a new window]   Table I. Calpain inhibitors did not inhibit adherence and intracellular survival of GBS in M

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Calpain activation during GBS-induced M apoptosis. A, Time course of calpain activity. M preincubated with 80 µM Suc-LLVY-AMC were infected with GBS and then fluorescence was measured over time. Calpain activity is shown as fold activation of calpain compared with activity in untreated controls. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M vs control M) according to Student’s t test. B, Effect of calpain and protease inhibitors on calpain activity. M pretreated in absence or presence of the indicated concentrations of inhibitors were preincubated with Suc-LLVY-AMC (80 µM) and then infected with GBS. Fluorescence was measured and histogram was generated from data time point at 2 h. Treating control M with inhibitors for the same times did not increase the fluorescence. Calpain activity is shown as fold activation of calpain compared with activity in untreated controls. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M treated with inhibitor vs untreated GBS-infected M) according to Student’s t test. C, The m-calpain activity in GBS-infected M. Cell lysates from control (C) and GBS-infected M, prepared at 2 h in PBS, were tested by casein zymography. A mixture of 0.1 µg each of commercial µ- and m-calpain was processed alongside and served as activity standard (std). Zymogram is representative of four independent experiments.

Bacterial interference in the enzymatic calpain assay was excluded because no enzymatic activity was detected in the bacterial preparation itself (data not shown).

To show that in particular the m-calpain activity is up-regulated during GBS-induced apoptosis we used casein zymography, a method useful for resolving both calpain isoforms (54, 55). The results in Fig. 4C show that m-calpain is activated in GBS-induced M apoptosis. In fact, while the proteolytic activity of µ-calpain was detected at low levels, at 2 h after infection, both in control and GBS-infected M, the proteolytic activity of m-calpain was detected only in GBS-infected M with levels similar to the band digested by standard m-calpain (Fig. 4C).

Because during GBS-induced M apoptosis we did not detect an increase in µ- and m-calpain expression levels by immunoblot analysis (data not shown), the above-reported calpain activation was at activity and not expression level.

Overall, the results indicate that calpains, in particular m-calpain, as a consequence of an extracellular Ca²⁺ influx, are activated and involved in GBS-induced M apoptosis.

Calpain-mediated Bax and Bid cleavage in GBS-induced M apoptosis

Calpains can be upstream mediators of apoptosis by triggering the mitochondrial death program through cleavage of some Bcl-2 family members (24, 25, 26, 40, 41, 42, 43), critical regulators of apoptotic mitochondrial pathways (21, 22, 23, 44, 45, 46).

We analyzed in GBS-induced M apoptosis whether calpains could cleave Bcl-2 family members. Immunoblot analysis showed differences only in the expression of Bax and Bid (Fig. 5A). GBS induced a strong reduction of f.l. Bax and Bid in M at 2 h after infection. At 12 h, f.l. Bax and Bid were no longer detectable (Fig. 5A). In contrast, Bcl-x_L remained unchanged all during the experiments (Fig. 5A). Though Bcl-2 was weakly detectable in M and thus could not be evaluated clearly in our model, it appeared unchanged during GBS-induced M apoptosis (Fig. 5A).

View larger version (78K): [in this window] [in a new window]   FIGURE 5. PD150606 and EGTA prevent Bax and Bid cleavage caused during GBS-induced M apoptosis. A, Time course of expression of some Bcl-2 family proteins. Whole-cell lysates from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE, probed with anti-Bax N-20, anti-Bid, anti-Bcl-xL, or anti-Bcl-2 Ab. -Actin was loading control. RAJI lysate was used as positive control for Bcl-2 family protein expression. B, Effect of PD150606 and EGTA on Bax and Bid cleavage. Whole-cell lysates from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with anti-Bax N-20 or anti-Bid Ab. -Actin was loading control. Blots in A and B are representative of four independent experiments.

Calpains can cleave f.l. Bax (p21) at the N terminus to generate an active fragment of 18 kDa (40, 41). Because the anti-Bax Ab used in the above experiments is specific for the N terminus, to determine whether the p18 Bax fragment was generated in GBS-induced apoptosis we used an anti-Bax Ab raised against f.l. mouse Bax. Results show the strong appearance of an 18-kDa Bax fragment (Fig. 6A) and generation of this cleavage product was prevented both by PD150606 and EGTA, which also prevented the decrease of p21 Bax (Fig. 6B), indicating that the decrease of f.l. Bax is due to generation of the p18 Bax cleavage product.

View larger version (59K): [in this window] [in a new window]   FIGURE 6. Bax is cleaved to a p18 fragment by calpains in GBS-induced M apoptosis. A, Time course of p18 Bax generation. Whole-cell lysates from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE and probed with anti-Bax B-9 mAb which detects both p21 and p18 Bax. -Actin was loading control. B, Effect of PD150606 and EGTA on p18 Bax generation. Whole-cell lysates from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with anti-Bax B-9 mAb. -Actin was loading control. Blots in A and B are representative of four independent experiments. Intact protein (solid arrow) and major fragment (open arrow) are indicated.

GBS causes mitochondrial apoptogenic protein release during M apoptosis induction

The results of calpain-mediated Bax and Bid cleavage suggest that a mitochondrial apoptotic pathway may be triggered in GBS-induced M apoptosis.

It is known that the next apoptotic pathway step after Bax and Bid activation is the release of apoptogenic proteins from mitochondria such as cyt c, implicated particularly in caspase-dependent apoptosis, and AIF and Endo G which elicit caspase-independent apoptotic pathways (21, 44, 45).

We found that cyt c is released into the cytosol during M apoptosis induction (Fig. 7A). Cytosolic cyt c accumulated significantly at 2 h after GBS infection and continued to increase up to 12 h, while there was no detectable cyt c in the cytosol of control M (Fig. 7A, left). Loss of mitochondrial cyt c was already evident at 2 h after infection and clearly apparent at 12 h (Fig. 7A, right).

View larger version (59K): [in this window] [in a new window]   FIGURE 7. In GBS-induced M apoptosis, AIF and cyt c are released into the cytosol. This effect is prevented by PD150606 and EGTA. A, cyt c and AIF release into cytosol. Cytosolic and mitochondrial subfractions from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE and probed with the appropriated Abs. -Actin was used as loading control for the cytosolic and Cox IV for the mitochondrial fraction. B, Effect of PD150606 and EGTA on cyt c and AIF release into cytosol. Cytosolic subfractions from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with the appropriated Abs. -Actin was used as loading control for the cytosolic fraction and Cox IV to evidence mitochondrial contamination. Blots in A and B are representative of four independent experiments.

Treatment of M both with PD150606 and EGTA abolished mitochondrial AIF and cyt c release into the cytosol already at 2 h (Fig. 7B).

To investigate the possible relationship between mitochondrial membrane permeabilization (MMP) induction and cyt c and AIF release, the effect of CsA, an inhibitor of PTP (44, 58), on cyt c and AIF release at 2 h was examined. CsA did not affect the release of cyt c or AIF, observed in GBS-induced M apoptosis (Fig. 8), nor apoptosis (data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 8. Effect of CsA on cyt c and AIF release in GBS-induced M apoptosis. Cytosolic subfractions from M untreated or pretreated with 10 µM CsA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with the appropriated Abs. -Actin was loading control for the cytosolic fraction and Cox IV to evidence mitochondrial contamination. Blots are representative of four independent experiments.

View larger version (63K): [in this window] [in a new window]   FIGURE 9. Analysis of mitochondrial Endo G in GBS-induced M apoptosis and effect of PD150606 and EGTA. A, Time course of mitochondrial Endo G level. Mitochondrial subfractions from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE and probed with anti-Endo G Ab. Cox IV was loading control for the mitochondrial fraction. B, Effect of PD150606 and EGTA on mitochondrial Endo G level. Mitochondrial subfractions from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with anti-Endo G Ab. Cox IV was loading control for the mitochondrial fraction. Blots in A and B are representative of three independent experiments.

The results suggest that in GBS-induced M apoptosis, mitochondrial protein release occurs and is controlled by calpain activation mediated by a Ca²⁺ increase and is not dependent on the opening of CsA-sensitive PTP.

Calpain-mediated cleavage of APAF-1, caspase-3 and -7 in GBS-induced M apoptosis

We previously demonstrated, using caspase inhibitors, that GBS-induced M apoptosis occurs in the absence of caspase activation (12).

The observation that, in GBS-induced apoptosis, cyt c was released from the mitochondria (Fig. 7A) led us to investigate why caspases were not activated. Because it is known that effector caspases and APAF-1 are degraded by calpains (32, 33, 34, 37, 38, 62), we looked for integrity and processing of caspase-3, -7, and APAF-1, in our apoptosis model. A strong reduction in f.l. APAF-1 was observed in GBS-induced M apoptosis (Fig. 10A) and the APAF-1 degradation was inhibited by PD150606 and EGTA (Fig. 10B) suggesting that calpains degrade APAF-1.

View larger version (42K): [in this window] [in a new window]   FIGURE 10. Cleavage of APAF-1, caspase-3 and -7 in GBS-induced M apoptosis mediated by calpains. Time course of APAF-1 (A) and caspase-3 and -7 (C) integrity and processing. Whole-cell lysates from control (C) and GBS-infected M, prepared at the indicated times, were subjected to SDS-PAGE and probed with the appropriated Abs. -Actin was loading control. Lysate from Jurkat cells treated with 0.25 mg/ml cyt c for 1 h was used as positive control for caspase-3 and -7 processing. Effect of PD150606 and EGTA on APAF-1 (B) and caspase-3 and -7 (D) degradation. Whole-cell lysates from M untreated, pretreated with 50 µM PD150606 or treated with 1 mM EGTA, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with the appropriated Abs. -Actin was loading control. Blots in this figure are representative of five independent experiments. Intact protein (solid arrow) and active fragment (open arrow) are indicated.

To further verify the absence of caspase-3 and -7 activation in our cell system, we assayed directly for caspase-3-like activity by measuring hydrolysis of the fluorogenic caspase-3-like protease peptide substrate Ac-DEVD-AMC (13, 39). We found no stimulation of the Ac-DEVD-AMC cleavage activity in GBS-infected M or in GBS-infected M pretreated with PD150606 compared with control M at all times examined (Table II), whereas exposure of M to 5 µM STS for 14 h (positive control for caspase-3 activation) (56, 60) caused a marked stimulation of caspase-3-like activity (a 40-fold increase) which was not induced when M were pretreated with 25 µM BAF before STS exposure (Table II). The experiments we performed with caspase-specific inhibitors, as expected, prevented cleavage of the substrate (Ac-DEVD-AMC), thus confirming the specificity for the changes observed in the caspase-3-like activity (data not shown).

View this table: [in this window] [in a new window]   Table II. Effect of GBS on caspase-3-like activity in M

Knockdown of m-calpain by siRNA inhibits GBS-induced M apoptosis

To provide nonpharmacological evidence of involvement of calpains in GBS-induced M apoptosis, we used the siRNA-mediated gene-silencing technique (63, 64, 65) to knockdown m-calpain and µ-calpain. Preliminary experiments with siRNA were performed to determine optimum dose and incubation time, examining siRNA concentration of 50, 100, and 200 nM for 24, 48, and 72 h. An siRNA concentration of 100 nM for 72 h exerted the maximum inhibition of mRNA and protein expression without affecting M viability (data not shown). Therefore M were transfected with 100 nM siRNA specific for µ-calpain or m-calpain for 72 h. Then at 72 h posttransfection, the cells were: 1) harvested for analysis of mRNA expression by qRT-PCR or protein expression by immunoblotting, 2) infected with GBS for apoptosis and immunoblot analysis. Transfection with m-calpain siRNA or µ-calpain siRNA optimally reduced mRNA (Fig. 11A) and protein levels (Fig. 11, B and C). In fact, qRT-PCR and immunoblotting demonstrated that mRNA and protein levels of m-calpain were inhibited by 75% and µ-calpain by 50% (Fig. 11). Mock transfection and transfection with 100 nM control nontargeting siRNA in M did not affect mRNA expression or protein levels of µ- or m-calpain (Fig. 11). Notably, neither µ-calpain siRNA or m-calpain siRNA affected the mRNA or protein levels of the other calpain isoform (Fig. 11). Down-regulation of m-calpain by m-calpain siRNA, but not of µ-calpain by µ-calpain siRNA, significantly prevented M apoptosis (65% inhibition) (Fig. 12A) and -spectrin cleavage (Fig. 12B) induced by GBS. Further knockdown of m-calpain reduced the degradation of Bax and Bid (Fig. 12C).

View larger version (29K): [in this window] [in a new window]   FIGURE 11. Knockdown of m-calpain or µ-calpain mRNA and protein levels by siRNA. Analysis of m-calpain and µ-calpain mRNA expression by qRT-PCR (A) or protein level by immunoblotting (B) following silencing of m-calpain or µ-calpain gene by siRNA. At 72 h posttransfection, cDNA or whole-cell lysates from M transfected, with vehicle alone (Mock), control nontargeting siRNA (100 nM; si control), m-calpain siRNA (100 nM; si m-calpain) or µ-calpain siRNA (100 nM; si µ-calpain) and nontransfected (NT), were subjected: to qRT-PCR and data were expressed as 2– Ct; or to immunoblotting with the anti-m-calpain or anti-µ-calpain Ab and anti--actin as loading control. Blots are representative of four independent experiments. C, Quantitative measurements of µ- or m-calpain protein bands was performed with Versadoc 1000 and Quantityone software. Data are expressed as percentage of NT M. Data in A and C are means ± SD of four independent experiments. *, p < 0.01 (M transfected with siRNA vs NT M) according to Student’s t test.

View larger version (41K): [in this window] [in a new window]   FIGURE 12. Silencing of m-calpain expression by siRNA inhibited GBS-induced M apoptosis, -spectrin cleavage, and Bax and Bid degradation. A, Effect of m-calpain siRNA and µ-calpain siRNA on apoptosis. At 72 h posttransfection, M transfected, with vehicle alone (Mock), control nontargeting siRNA (100 nM; si control), µ-calpain siRNA (100 nM; si µ-calpain) and m-calpain siRNA (100 nM; si m-calpain) or nontransfected (NT), were infected with GBS for 2 h and then recovered at 24 h, and apoptosis was measured by evaluating the percentage of hypodiploid nuclei at flow cytometry. There was no apoptosis in M transfected but not infected. Data are means ± SD of four experiments done in triplicate. *, p < 0.01 (GBS-infected M transfected with m-calpain siRNA vs NT GBS-infected M) according to Student’s t test. Effect of m-calpain siRNA on -spectrin cleavage (B) and Bax and Bid degradation (C). Whole-cell lysates from M transfected, with µ-calpain siRNA (100 nM; si µ-calpain), m-calpain siRNA (100 nM; si m-calpain), and NT, infected or not with GBS, prepared at 2 h, were subjected to SDS-PAGE and probed with the appropriate Abs. -Actin was loading control. Blots are representative of four independent experiments. Intact protein (solid arrow) and active fragment (open arrow) are indicated.

These results, supporting the data obtained using pharmacological calpain inhibitors, provide further evidence that calpains, particularly m-calpain, are involved in GBS-induced M apoptosis.

Correlation of proapoptotic mediator degradations with GBS-induced M apoptosis

It is known that bacteria are able to activate/inactivate proapoptotic and antiapoptotic proteins such as caspases and Bcl-2 family members, to modulate the normal functions of host cell apoptotic pathways (18, 19, 20). Furthermore, there is evidence showing that during infection the caspases and Bcl-2 family proteins are activated without participating in apoptosis (18, 19, 20).

To assess whether the degradation of Bax, Bid, APAF-1, and caspases occurring during GBS infection is relevant for GBS-induced M apoptosis or occurs as a consequence of GBS infection, we performed experiments using gGBS, which during infection does not induce M apoptosis (Ref. 12 , and Fig. 13A), having lost its hemolysin expression (12).

View larger version (31K): [in this window] [in a new window]   FIGURE 13. Effect of gGBS infection on proapoptotic mediator degradations. A, Apoptosis in control (C), GBS-infected M and gGBS-infected M, was measured at 24 h evaluating the percentage of hypodiploid nuclei at flow cytometry. Data are means ± SD of six experiments done in triplicate. *, p < 0.01 (GBS-infected M vs control M) according to Student’s t test. B, Bax, Bid, APAF-1, caspase-3 and -7 degradation during gGBS infection. Whole-cell lysates from control (C), GBS-infected M and gGBS-infected M, prepared at 2 h, were subjected to SDS-PAGE and probed with the appropriate Abs. -Actin was loading control. Blots are representative of three independent experiments.

These results showing that infection with gGBS, which did not induce apoptosis, did not cause degradation of the above-mentioned proapoptotic mediators suggest that Bax, Bid, APAF-1, caspase-3 and -7 degradation is correlated with apoptosis induction and not an effect of GBS infection.

To further assess whether Bax and Bid degradation is correlated to GBS-induced M apoptosis, we used Bax siRNA and Bid siRNA to inhibit Bax and Bid expression, respectively. For this, M were transfected with 100 nM Bax siRNA or with 100 nM Bid siRNA. At 72 h posttrasfection, the cells were harvested for both mRNA and protein expression analysis or infected with GBS for apoptosis analysis. We found that transfection of M with Bax siRNA resulted in an 35% decrease of mRNA and protein level of Bax compared with nontransfected M (Fig. 14, A–C) but we did not observe any effect on Bid mRNA and protein levels (Fig. 14, A–C). As shown in Fig. 14D, down-regulation of Bax resulted in 30% reduction of GBS-induced M apoptosis.

View larger version (32K): [in this window] [in a new window]   FIGURE 14. Silencing Bax or Bid expression by siRNA decreases GBS-induced M apoptosis. Analysis of Bax and Bid mRNA by qRT-PCR (A) or protein level by immunoblotting (B) following silencing of Bax or Bid gene by siRNA. At 72 h posttransfection, cDNA or whole-cell lysates from M transfected, with vehicle alone (Mock), control nontargeting siRNA (100 nM; si control), Bax siRNA (100 nM; si Bax) or Bid siRNA (100 nM; si Bid) and nontransfected (NT), were subjected: to qRT-PCR and data expressed as 2– Ct; or to immunoblotting with the anti-Bax or anti-Bid Ab and anti--actin as loading control. Blots are representative of four independent experiments. C, Quantitative measurements of Bax or Bid protein bands was performed with Versadoc 1000 and Quantityone software. Data are expressed as percentage of NT M. Data in A and C are means ± SD of four independent experiments. *, p < 0.01 (M transfected with siRNA vs NT M) according to Student’s t test. D, Effect of Bax siRNA or Bid siRNA on apoptosis. At 72 h posttransfection, M transfected with vehicle alone (Mock), control nontargeting siRNA (100 nM; si control), Bax siRNA (100 nM; si Bax) and Bid siRNA (100 nM; si Bid) or nontransfected (NT), were infected with GBS for 2 h then recovered at 24 h and apoptosis measured evaluating the percentage of hypodiploid nuclei at flow cytometry. There was no apoptosis in M transfected but not infected. Data are means ± SD of four experiments done in triplicate. *, p < 0.01 (GBS-infected M transfected with siRNA vs NT GBS-infected M) according to Student’s t test.

Mock transfection and transfection with control nontargeting siRNA in M did not affect Bax or Bid mRNA and protein expression (Fig. 14, A–C) or apoptosis (Fig. 14D).

The results of Bax and Bid knockdown suggest that Bax and Bid degradation is correlated to apoptosis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We have demonstrated that GBS induces M apoptosis which is countered by inhibition of extracellular Ca²⁺ influx but is not affected by inhibition of caspases (12). Because pathogens can induce apoptosis in immune cells through distinct pathways leading to different functional or pathological consequences (16, 17, 18, 19, 20), this study analyzed the mechanisms by which GBS induces apoptosis in M.

Our results show that GBS activates calpains, as a consequence of intracellular Ca²⁺ increase, and calpains play a central role in GBS-induced M apoptosis initiating a mitochondrial-dependent but caspase-independent apoptotic program.

GBS caused a strong intracellular Ca²⁺ increase reaching millimolar concentration and with kinetics like the GBS-induced M membrane permeability defects which we previously demonstrated to be strictly correlated to apoptosis induction (12). On the contrary, hi-GBS, which did not induce M membrane permeability defects and apoptosis, did not cause [Ca²⁺]_i increase. These observations and the demonstration that EGTA (Ref. 12 and this study) inhibits GBS-induced M apoptosis indicate that the [Ca²⁺]_i increase may be due to an extracellular Ca²⁺ influx consequent to membrane permeability defects and point to a strong connection between Ca²⁺ increase and apoptosis.

As a consequence of GBS-induced Ca²⁺ increase, the Ca²⁺-dependent protease calpains are activated as demonstrated by: 1) the cleavage of the calpain substrate -spectrin into a 145-kDa BP, in the absence of caspase activity, 2) the increased total levels of calpain activity measured as cleavage of the fluorogenic calpain substrate Suc-LLVY-AMC both in situ and in cell extracts, 3) the prevention of -spectrin and Suc-LLVY-AMC proteolysis by EGTA (Ca²⁺ chelator), and µ- and m-calpain inhibitors directed to the Ca²⁺-binding site (PD150606) or to the active site (CS peptide, leupeptin, MDL28170, ALLN) but not by PD151746 (µ-calpain inhibitor) and other protease inhibitors, and 4) the casein zymography. Our results concur with many reports suggesting that various stimuli that increase cytosolic Ca²⁺ trigger an apoptotic program mediated by calpains (27, 28, 29, 30, 31, 32, 33, 34, 35, 58). These findings also rule out that, similar to what occurs in some apoptotic models, calpain activation may require other factors such as phospholipids, intracellular redistribution of calpains, and caspase-mediated degradation of the endogenous inhibitor calpastatin (29, 36, 43).

Calpains are essential for induction of M apoptosis by GBS because several µ- and m-calpain inhibitors such as PD150606, CS peptide, leupeptin, MDL28170, and ALLN inhibit apoptosis while CA-074 Me (cathepsin B inhibitor), NH₄Cl (inhibitor of all lysosomal cathepsins), BAF (caspase inhibitor), and clasto-lactacystin (proteasome inhibitor) do not. In particular, m-calpains were up-regulated by GBS and seemed to have a major role because casein zymography, an assay which distinguishes the activity of both calpain isoforms (54, 55), revealed a strong m-calpain activity only in GBS-infected M, and PD151746, a µ-calpain inhibitor, did not affect GBS-induced apoptosis. The involvement of calpains, particularly m-calpain, in GBS-induced M apoptosis is also supported by nonpharmacological inhibition of calpain activity obtained with calpain-specific siRNA-mediated gene silencing. In fact, we found that knockdown of m-calpain but not of µ-calpain significantly inhibited GBS-induced M apoptosis and -spectrin proteolysis.

In an attempt to define the mechanisms by which activated calpains induced apoptosis in our model, we examined their role in the cleavage of Bcl-2 family proteins. It is known that several proteases, including calpains, cleave and activate Bid and Bax in different apoptotic models (Refs. 21, 22, 23, 24, 25, 26 , 34 , and 40, 41, 42, 43). Because Bid and Bax play a key role in inducing MMP, which in turn leads to release of apoptogenic mitochondrial factors, activation of Bid and Bax, either occurring transcriptionally or by conformational changes, induced by cleavage or binding to an activated "BH3 domain-only" member of the Bcl-2 family, is important for apoptosis. This study shows that Bax and Bid are cleaved during GBS-induced M apoptosis and the calpains are responsible for proteolysis. In fact, protection against cleavage of Bid and Bax was obtained with PD150606 and m-calpain siRNA but not with cathepsin B inhibitor CA-074 Me or the caspase inhibitor BAF (data not shown). The absence of Bcl-x_L and Bcl-2 cleavage suggests that calpains specifically cleave Bax and Bid and the effect is not due to uncontrolled calpain-mediated proteolysis. In GBS-induced M apoptosis, p21 Bax is converted by calpains into a p18 Bax-truncated form, consistent with evidence that calpains like other proteases cleave Bax to a p18 fragment (40, 41). However, it remains to be defined whether in our apoptosis model this proteolysis enhanced the apoptogenic properties of Bax, as described in the literature (41, 66).

Although it is known that MMP may be controlled by Bcl-2 family proteins (e.g., Bax) the exact molecular mechanisms of MMP are still a matter of debate (44, 45, 46). It is suggested that proapoptotic Bcl-2 family members can act both as crucial pore-forming molecules leading to mitochondrial outer membrane permeabilization (MOMP) and as components of PTP complex which constitutes a megachannel. Therefore, proapoptotic Bcl-2 members may cause release of apoptotic mitochondrial factors by induction of MOMP or by PTP (44, 45, 46). There is also evidence that Ca²⁺ may cause direct mitochondrial damage leading to PTP (24, 44, 58). In GBS-induced M apoptosis, we found that calpain inhibition, which blocks Bax and Bid cleavage, prevents mitochondrial cyt c and AIF release while CsA, an inhibitor of PTP (44, 58), did not affect M apoptosis, cyt c or AIF release. This evidence, even if the molecular mechanism of AIF and cyt c release in GBS-induced M apoptosis is not clear, suggests that these mitochondrial factors are released as a consequence of MMP induced by Bax and Bid activation mediated by calpains but this release does not involve opening of PTP. These findings also rule out the possibility that in GBS-induced apoptosis mitochondrial factor release may occur as a result of the direct effect of Ca²⁺ increase on the mitochondria.

Cytosolic cyt c increase is associated with activation of effector caspases. In fact, cyt c promotes assembly of APAF-1 and procaspase-9 in the apoptosome, which containing active caspase-9, can process proteolitically caspase-3 and -7 (21, 22, 23, 45, 46). In GBS-induced M apoptosis although cyt c is released, there was no evidence of caspase-3 and -7 activation. The inability of released cyt c to activate caspases in GBS-induced apoptosis seems due to calpain-mediated degradation of APAF-1 (a necessary cofactor), and of caspase-3 and -7, as described for other calpains- or Ca²⁺-mediated apoptotic models (32, 33, 34, 37, 38, 62, 67). However, because calpain inhibition prevented cyt c release, caspase activation did not occur even following calpain inhibition by PD150606 which abolished APAF-1 and caspase degradation. This finding further supports that in our model, GBS-induced M apoptosis is dependent on calpain activation but is caspase independent.

Our results also indicate that the molecular mechanism of GBS-induced apoptosis differs from caspase-dependent apoptosis mechanisms described for the majority of pathogens. Caspase activation was observed in apoptosis induced: by intracellular pathogens which interact directly with components of the apoptotic pathways, by extracellular pathogens that indirectly induce the apoptotic pathways by translocation of bacterial toxins into the host cells and by Neisseria which causes extracellular Ca²⁺ influx and calpain activation (17, 18, 19, 20, 68, 69). Instead, GBS only activates calpains.

The cyt c is not the only proapoptotic mitochondrial protein playing a role in apoptosis induction (21, 22, 23, 45, 46). AIF and Endo G have also been identified as proteins with an important proapoptotic function in several caspase-independent apoptosis (24, 25, 26, 45, 46, 47, 48, 49). In particular, it has been demonstrated that AIF is a main executioner of Pneumococcus-induced apoptosis where despite cyt c release caspases were weakly activated (70, 71). Therefore, because during GBS-induced M apoptosis AIF is released, in our system, the death pathway, initiated downstream of mitochondria, appears to be mainly mediated by AIF. This seems in contrast with our previous demonstration (12) that dying GBS-infected M, despite lack of caspase activation, present oligonucleosomal DNA fragmentation (200 bp), because the apoptotic function of AIF is the formation of large chromatin fragments (50 kbp) (Refs. 24, 25, 26 , 28 , and 45, 46, 47, 48, 49). However, because there is a weak release of Endo G from mitochondria in the cells of our system, AIF is probably assisted by Endo G, which causes caspase-independent oligonucleosomal DNA fragmentation (45), to promote efficient DNA degradation in GBS-induced M apoptosis. These results suggest that the importance of Bax activation in our model may be due to triggering of mitochondrial caspase-independent apoptotic pathways, initiated by AIF and Endo G release. Regarding cleavage of Bid in GBS-induced M apoptosis, because we were unable to detect activated p15 Bid, it is still to be defined how Bid exerts its proapoptotic function in our model.

However, although it is known that during infections the caspases and Bcl-2 family proteins can be activated/inactivated without participating in apoptosis (18, 19, 20), in our model the degradation of proapoptotic mediators is correlated with M apoptosis induction and is not an effect of GBS infection because gGBS, which did not induce apoptosis (Ref. 12 and this study), did not cause degradation of the Bax, Bid, APAF-1, caspase-3 and -7. The correlation of Bax and Bid degradation with GBS-induced M apoptosis was also suggested by using Bax siRNA and Bid siRNA. In fact, although when using Bax siRNA and Bid siRNA, we obtained only 35% down-regulation of Bax and Bid expression, respectively, there was a reduction of GBS-induced M apoptosis.

In a recent study by Ulett et al. (60), it was demonstrated, unlike what is shown in this study, that GBS-induced M apoptosis is caspase-3 dependent and GBS provokes unique changes in Bad, 14-3-3, and Omi/HtrA2. However, in the Ulett apoptotic model GBS induced a 30% M apoptosis at 48 h and 70% only after 96 h (13, 60), while in our model GBS caused 80% M apoptosis at 24 h (Ref. 12 and this study). There was no early alteration of the M plasma membrane permeability with influx of Ca²⁺ and no calpain activation in their model. In our model, there were after 2 h. Furthermore, they used GBS type III strain 874391, J774 M-like cell line, and thioglycolate-elicited M from C57BL/6 mice (13, 60). In our model, we used GBS type III strain COH 31 r/s and thioglycolate-elicited M from CD1 mice. Comparing the two models, it seems that GBS can induce early (24 h) and late (48 h) M apoptosis, and the apoptosis which occurs at 24 h is characterized by alterations in plasma membrane permeability, influx of extracellular Ca²⁺, and, as shown in this study, calpain activation, while late apoptosis described in the Ulett et al. model (60) does not show these features. Therefore, these key differences between the two models may explain the different molecular mechanisms described.

Furthermore, as regards the GBS factor responsible for initiating apoptosis, our model presents different characteristics from that of Ulett et al. (13). In fact, we showed in this and our previous work that gGBS, having lost -hemolysin, did not induce M apoptosis at 24 h while in the Ulett et al. model (13), the -hemolysin-deficient isogenic mutant of GBS still induced M apoptosis. However, this occurred at 48 h whereas we evaluated the effect of gGBS at 24 h the time of maximum apoptosis induction for the -hemolytic GBS in our system.

Another report, by Liu et al. (72), showed both that the -hemolysin-deficient isogenic mutant of the GBS strain V and Ia do not induce M apoptosis at 24 h, and that the ability of -hemolytic wild-type parent GBS strain to induce massive M apoptosis at 24 h depends on the -hemolytic titer. This suggests that GBS -hemolysin is involved in induction of M apoptosis at 24 h (72). Altogether, these findings suggest that GBS can induce M apoptosis with different apoptotic inducing characteristics, early, in a -hemolysin-dependent manner (Refs. 12 and 72 , and this study) and in a -hemolysin-independent manner (13, 60) which is mediated by a factor not yet defined, further confirming two different models of M apoptosis induction by GBS underlying diverse molecular mechanisms.

Therefore, it is likely that the relative expression of these two factors on the GBS affect the inducing characteristics of apoptosis. Further studies on the apoptotic mechanisms of different GBS strains and on the different susceptibility of diverse cell types, both immune and nonimmune to apoptosis may provide more revealing information.

In conclusion, this study provides the first evidence that GBS induces M apoptosis through an extracellular Ca²⁺ influx and the activation of calpains, particularly m-calpain, which then trigger a caspase-independent mitochondrial pathway. This apoptotic pathway used by GBS seems unusual for the mechanisms of bacterial-induced apoptosis where, with only few exceptions, caspase activation is essential (18, 19, 20, 68, 69, 70, 71). Therefore, the ability of GBS to induce apoptosis in M, essential APC and effector cells of immune responses, by this calpain-dependent and caspase-independent mechanism could be an important pathogenic mechanism by which GBS evade host immune responses and cause disease.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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 a grant from Fondazione Cassa di Risparmio di Perugia, Italy (2002 and 2005) and Polo Didattico e Scientifico di Terni, Italy (2005).

² Address correspondence and reprint requests to Prof. Pierfrancesco Marconi, Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, University of Perugia, General Hospital, Monteluce, 06100 Perugia, Italy. E-mail address: marimmun{at}unipg.it

³ Abbreviations used in this paper: GBS, Group B Streptococcus; M, macrophage; cyt c, cytochrome c; AIF, apoptosis-inducing factor; Endo G, endonuclease G; PTP, permeability transition pore; CsA, cyclosporin A; STS, staurosporine; PI, propidium iodide; siRNA, small-interfering RNA; hi-GBS, heat-inactivated GBS; gGBS, GBS grown in the presence of glucose; [Ca²⁺]_i, intracellular Ca²⁺ concentration; qRT-PCR, quantitative RT-PCR; Ct, cycle threshold; BP, breakdown product; f.l., full length; MMP, mitochondrial membrane permeabilization; MOMP, mitochondrial outer membrane permeabilization; PD150606, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid; PD151746, 3-(5-fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid; PD145305, 2-mercapto-3-phenylpropanonic acid; CS peptide, 27-residue calpastatin peptide; MDL28170, Carbobenzoxy-valinyl-phenylalaninal; ALLN, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; CA-074 Me, cathepsin B inhibitor IV; BAF, Boc-Asp(OMe)-fluoromethylketone; clasto-lactacystin, clasto-lactacystin -Lactone; Cox IV, cyt c oxidase subunit IV; Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; M, macrophage.

Received for publication November 17, 2005. Accepted for publication April 4, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Baker, C. J., M. S. Edwards. 1995. Group B streptococcal infections. J. Remington, and J. O. Klein, eds. Infectious Diseases of the Fetus and Newborn Infant 4th Ed.980-1054. W. B. Saunders, Philadelphia.
2. Rubens, C. E., M. R. Wessel, L. M. Heggen, D. L. Kasper. 1987. Transposon mutagenesis of type III group B streptococcus: correlation of capsule expression with virulence. Proc. Natl. Acad. Sci. USA 84: 7208-7212. [Abstract/Free Full Text]
3. Orefici, G., S. Recchia, L. Galante. 1988. Possible virulence marker for Streptococcus agalactiae (Lancefield Group B). Eur. J. Clin. Microbiol. Infect. Dis. 7: 302-305. [Medline]
4. Hulse, M. L., S. Smith, E. Y. Chi, A. Pham, C. E. Rubens. 1993. Effect of type III group B streptococcal capsular polysaccharide on invasion of respiratory epithelial cells. Infect. Immun. 61: 4835-4841. [Abstract/Free Full Text]
5. Rubens, C. E., S. Smith, M. L. Hulse, E. Y. Chi, G. Van Belle. 1992. Respiratory epithelial cell invasion by group B streptococci. Infect. Immun. 60: 5157-5163. [Abstract/Free Full Text]
6. Gibson, R. L., M. K. Lee, C. Soderland, E. Y. Chi, C. E. Rubens. 1993. Group B streptococcal invade endothelial cells: type III capsular polysaccharide attenuates invasion. Infect. Immun. 61: 478-485. [Abstract/Free Full Text]
7. Valentin-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. [Abstract]
8. 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. [Medline]
9. Tapsall, J. W., E. A. Phillips. 1991. The hemolytic and cytolytic activity of group B streptococcal hemolysin and its possible role in early onset group B streptococcal disease. Pathology 23: 139-144. [Medline]
10. Nizet, V., R. L. Gibson, E. Y. Chi, P. E. Framson, M. L. Hulse, C. E. Rubens. 1996. Group B streptococcal -hemolysin expression is associated with injury of lung epithelial cells. Infect. Immun. 64: 3818-3826. [Abstract]
11. Nizet, V., K. S. Kim, M. Stins, M. Jonas, E. Y. Chi, D. Nguyen, C. E. Rubens. 1997. Invasion of brain microvascular endothelial cells by group B streptococci. Infect. Immun. 65: 5074-5081. [Abstract]
12. 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. [Abstract/Free Full Text]
13. Ulett, G. C., J. F. Bohnsack, J. Armstrong, E. E. Adderson. 2003. -Hemolysin-independent induction of apoptosis of macrophages infected with serotype III group B streptococcus. J. Infect. Dis. 188: 1049-1053. [Medline]
14. Ring, A., J. S. Braun, J. Pohl, V. Nizet, W. Stremmel, J. L. Shenep. 2002. Group B streptococcal -hemolysin induces mortality and liver injury in experimental sepsis. J. Infect. Dis. 185: 1745-1753. [Medline]
15. Leib, S. L., Y. S. Kim, L. L. Chow, R. A. Sheldon, M. G. Tauber. 1996. Reactive oxygen intermediates contribute to necrotic and apoptotic neuronal injury in an infant rat model of bacterial meningitis due to group B streptococci. J. Clin. Invest. 98: 2632-2639. [Medline]
16. Weinrauch, Y., A. Zychlinsky. 1999. The induction of apoptosis by bacterial pathogens. Annu. Rev. Microbiol. 53: 155-187. [Medline]
17. Navarre, W. W., A. Zychlinsky. 2000. Pathogen-induced apoptosis of macrophages: a common end for different pathogenic strategies. Cell Microbiol. 2: 265-273. [Medline]
18. Gao, L. Y., Y. A. Kwaik. 2000. The modulation of host cell apoptosis by intracellular bacterial pathogens. Trends Microbiol. 8: 306-313. [Medline]
19. Grassme, H., V. Jendrossek, E. Gulbins. 2001. Molecular mechanisms of bacteria induced apoptosis. Apoptosis 6: 441-445. [Medline]
20. Gao, L., Y. Abu Kwaik. 2000. Hijacking of apoptotic pathways by bacterial pathogens. Microbes Infect. 2: 1705-1719. [Medline]
21. Kaufmann, S. H., M. O. Hengartner. 2001. Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 11: 526-534. [Medline]
22. Strasser, A., L. O’Connor, V. M. Dixit. 2000. Apoptosis signaling. Annu. Rev. Biochem. 69: 217-245. [Medline]
23. Budihardjo, I., H. Oliver, M. Lutter, X. Luo, X. Wang. 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell. Dev. Biol. 15: 269-290. [Medline]
24. Jaattela, M., J. Tschopp. 2003. Caspase-independent cell death in T lymphocytes. Nat. Immunol. 4: 416-423. [Medline]
25. Mathiasen, I. S., M. Jaattela. 2002. Triggering caspase-independent cell death to combat cancer. Trends Mol. Med. 8: 212-220. [Medline]
26. Bidere, N., A. Senik. 2001. Caspase-independent apoptotic pathways in T lymphocytes: a minireview. Apoptosis 6: 371-375. [Medline]
27. Squier, M. K., J. J. Cohen. 1997. Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158: 3690-3697. [Abstract]
28. Mathiasen, I. S., I. N. Sergeev, L. Bastholm, F. Elling, A. W. Norman, M. Jaattela. 2002. Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells. J. Biol. Chem. 277: 30738-30745. [Abstract/Free Full Text]
29. Squier, M. K., A. J. Sehnert, K. S. Sellins, A. M. Malkinson, E. Takano, J. J. Cohen. 1999. Calpain and calpastatin regulate neutrophil apoptosis. J. Cell Physiol. 178: 311-319. [Medline]
30. Debiasi, R. L., M. K. Squier, B. Pike, M. Wynes, T. S. Dermody, J. J. Cohen, K. L. Tyler. 1999. Reovirus-induced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors. J. Virol. 73: 695-701. [Abstract/Free Full Text]
31. Ray, S. K., M. Fidan, M. W. Nowak, G. G. Wilford, E. L. Hogan, N. L. Banik. 2000. Oxidative stress and Ca²⁺ influx upregulate calpain and induce apoptosis in PC12 cells. Brain Res. 852: 326-334. [Medline]
32. Lankiewicz, S., C. M. Luetjens, N. Truc Bui, A. J. Krohn, M. Poppe, G. M. Cole, T. C. Saido, J. H. Prehn. 2000. Activation of calpain I converts excitotoxic neuron death into a caspase independent cell death. J. Biol. Chem. 275: 17064-17071. [Abstract/Free Full Text]
33. Wolf, B. B., J. C. Goldstein, H. R. Stennicke, H. Beere, G. P. Amarante-Mendes, G. S. Salvesen, D. R. Green. 1999. Calpain functions in a caspase independent manner to promote apoptosis-like events during platelet activation. Blood 94: 1683-1692. [Abstract/Free Full Text]
34. Gil-Parrado, S., A. Fernandez-Montalvan, I. Assfalg-Machleidt, O. Popp, F. Bestvater, A. Holloschi, T. A. Knoch, E. A. Auerswald, K. Welsh, J. C. Reed, et al 2002. Ionomycin-activated calpain triggers apoptosis: a probable role for Bcl-2 family members. J. Biol. Chem. 277: 27217-27226. [Abstract/Free Full Text]
35. Porn-Ares, M. I., T. C. Saido, T. Andersson, M. P. Ares. 2003. Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells. Biochem. J. 374: 403-411. [Medline]
36. Carafoli, E., M. Molinari. 1998. Calpain a protease in search of a function?. Biochem. Biophys. Res. Commun. 247: 193-203. [Medline]
37. McGinnis, K. M., M. E. Gnegy, Y. H. Park, N. Mukerjee, K. K. W. Wang. 1999. Procaspase 3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem. Biophys. Res. Commun. 263: 94-99. [Medline]
38. Chua, B. T., K. Guo, P. Li. 2000. Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J. Biol. Chem. 275: 5131-5135. [Abstract/Free Full Text]
39. Nath, R., K. J. Raser, D. Stafford, I. Hajimohammadreza, A. Posner, H. Allen, R. V. Talanian, P. Yuen, R. B. Gilbertsen, K. K. Wang. 1996. Non-erythroid -spectrin breakdown by calpain and interleukin 1-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319: 683-690. [Medline]
40. Wood, D. E., A. Thomas, L. A. Devi, Y. Berman, R. C. Beavis, J. C. Reed, E. W. Newcomb. 1998. Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17: 1069-1078. [Medline]
41. Gao, G., Q. P. Dou. 2000. N-terminal cleavage of bax by calpain generates a potent proapoptotic 18 kDa fragment that promotes bcl-2-independent cytochrome c release and apoptotic cell death. J. Cell Biochem. 80: 53-72. [Medline]
42. Chen, M., H. He, S. Zhan, S. Krajewski, J. C. Reed, R. A. Gottlieb. 2001. Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J. Biol. Chem. 276: 30724-30728. [Abstract/Free Full Text]
43. Mandic, A., K. Viktorsson, L. Strandberg, T. Heiden, J. Hansson, S. Linder, M. C. Shoshan. 2002. Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis. Mol. Cell Biol. 22: 3003-3013. [Abstract/Free Full Text]
44. Parone, P. A., D. James, J. C. Martinou. 2002. Mitochondria: regulating the inevitable. Biochimie 84: 105-111. [Medline]
45. Ravagnan, L., T. Roumier, G. Kroemer. 2002. Mitochondria-the killer organelles and their weapons. J. Cell Physiol. 192: 131-137. [Medline]
46. van Loo, G., X. Saelens, M. van Gurp, M. MacFarlane, S. J. Martin, P. Vandenabeele. 2002. The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ. 9: 1031-1042. [Medline]
47. Daugas, E., S. A. Susin, N. Zamzami, K. F. Ferri, T. Irinopoulou, N. Larochette, M. C. Prevost, B. Leber, D. Andrews, J. Penninger, G. Kroemer. 2000. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14: 729-739. [Abstract/Free Full Text]
48. Cande, C., F. Cecconi, P. Dessen, G. Kroemer. 2002. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death?. J. Cell Sci. 115: 4727-4734. [Medline]
49. Cande, C., I. Cohen, E. Daugas, L. Ravagnan, N. Larochette, N. Zamzami, G. Kroemer. 2002. Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie 84: 215-222. [Medline]
50. Ruckdeschel, K., A. Roggenkamp, V. Lafont, P. Mangeat, J. Heesemann, B. Rouot. 1997. Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis. Infect. Immun. 65: 4813-4821. [Abstract]
51. Bronk, S. F., G. J. Gores. 1993. pH-Dependent nonlysosomal proteolysis contributes to lethal anoxic injury of rat hepatocytes. Am. J. Physiol. 264: G744-G751. [Medline]
52. Chen, M., H. L. Fernandez. 2004. Stimulation of -amyloid precursor protein -processing by phorbol ester involves calcium and calpain activation. Biochem. Biophys. Res. Commun. 316: 332-340. [Medline]
53. Kohli, V., W. Gao, C. A. Camargo, P. A. Clavien. 1997. Calpain is a mediator of preservation-reperfusion injury in rat liver transplantation. Proc. Natl. Acad. Sci. USA 94: 9354-9359. [Abstract/Free Full Text]
54. Raser, K. J., A. Posner, K. K. Wang. 1995. Casein zymography: a method to study µ-calpain, m-calpain, and their inhibitory agents. Arch. Biochem. Biophys. 319: 211-216. [Medline]
55. Witkowski, J. M., E. Z. Trzebiatowska, J. M. Swiercz, M. Cichorek, H. Ciepluch, K. Lewandowski, E. Bryl, A. Hellmann. 2002. Modulation of the activity of calcium-activated neutral proteases (calpains) in chronic lymphocytic leukemia (B-CLL) cells. Blood 100: 1802-1809. [Abstract/Free Full Text]
56. Liu, J., D. P. Thewke, Y. R. Su, M. F. Linton, S. Fazio, M. S. Sinensky. 2005. Reduced macrophage apoptosis is associated with accelerated atherosclerosis in low-density lipoprotein receptor-null mice. Arterioscler. Thromb. Vasc. Biol. 25: 174-179. [Abstract/Free Full Text]
57. Heid, C. A., J. Stevens, K. J. Livak, P. M. Williams. 1996. Real time quantitative PCR. Genome Res. 6: 986-994. [Abstract/Free Full Text]
58. Hajnoczky, G., E. Davies, M. Madesh. 2003. Calcium signaling and apoptosis. Biochem. Biophys. Res. Commun. 304: 445-454. [Medline]
59. Donkor, I. O.. 2000. A survey of calpain inhibitors. Curr. Med. Chem. 7: 1171-1188. [Medline]
60. Ulett, G. C., K. H. Maclean, S. Nekkalapu, J. L. Cleveland, E. E. Adderson. 2005. Mechanisms of group B streptococcal-induced apoptosis of murine macrophages. J. Immunol. 175: 2555-2562. [Abstract/Free Full Text]
61. Paolini, R., R. Molfetta, M. Piccoli, L. Frati, A. Santoni. 2001. Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement. Proc. Natl. Acad. Sci. USA 98: 9611-9616. [Abstract/Free Full Text]
62. McGinnis, K. M., M. E. Gnegy, N. Falk, R. Nath, K. K. Wang. 2003. Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SY5Y neuroblastoma cells. Neurochem. Int. 42: 517-523. [Medline]
63. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498. [Medline]
64. McManus, M. T., P. A. Sharp. 2002. Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3: 737-747. [Medline]
65. Galli, A., G. Bergamaschi, H. Recalde, G. Biasiotto, P. Santambrogio, S. Boggi, S. Levi, P. Arosio, M. Cazzola. 2004. Ferroportin gene silencing induces iron retention and enhances ferritin synthesis in human macrophages. Br. J. Haematol. 127: 598-603. [Medline]
66. Wood, D. E., E. W. Newcomb. 2000. Cleavage of Bax enhances its cell death function. Exp. Cell Res. 256: 375-382. [Medline]
67. Reimertz, C., D. Kogel, S. Lankiewicz, M. Poppe, J. H. Prehn. 2001. Ca²⁺-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1). J. Neurochem. 78: 1256-1266. [Medline]
68. Nakagawa, I., M. Nakata, S. Kawabata, S. Hamada. 2001. Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells. Cell Microbiol. 3: 395-405. [Medline]
69. Muller, A., D. Gunther, F. Dux, M. Naumann, T. F. Meyer, T. Rudel. 1999. Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases. EMBO J. 18: 339-352. [Medline]
70. Braun, J. S., R. Novak, P. J. Murray, C. M. Eischen, S. A. Susin, G. Kroemer, A. Halle, J. R. Weber, E. I. Tuomanen, J. L. Cleveland. 2001. Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus. J. Infect. Dis. 184: 1300-1309. [Medline]
71. Braun, J. S., J. E. Sublett, D. Freyer, T. J. Mitchell, J. L. Cleveland, E. I. Tuomanen, J. R. Weber. 2002. Pneumococcal pneumolysin and H₂O₂ mediate brain cell apoptosis during meningitis. J. Clin. Invest. 109: 19-27. [Medline]
72. Liu, G. Y., K. S. Doran, T. Lawrence, N. Turkson, M. Puliti, L. Tissi, V. Nizet. 2004. Sword and shield: linked group B streptococcal -hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proc. Natl. Acad. Sci. USA 101: 14491-14496. [Abstract/Free Full Text]

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