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alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling.

Bantel H, Sinha B, Domschke W, Peters G, Schulze-Osthoff K, Jänicke RU - J. Cell Biol. (2001)

Bottom Line: Furthermore, alpha-toxin-induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2.Together with our finding that alpha-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus alpha-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors.Hence, our findings clearly define a signaling pathway used in S. aureus-induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Cell Biology, University of Münster, 48149 Münster, Germany.

ABSTRACT
Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and DNA fragmentation were induced not only when Jurkat T cells were infected with intact bacteria, but also after treatment with supernatants of various S. aureus strains. We also demonstrate that S. aureus-induced cell death and caspase activation were mediated by alpha-toxin, a major cytotoxin of S. aureus, since both events were abrogated by two different anti-alpha-toxin antibodies and could not be induced with supernatants of an alpha-toxin-deficient S. aureus strain. Furthermore, alpha-toxin-induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2. Together with our finding that alpha-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus alpha-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors. Hence, our findings clearly define a signaling pathway used in S. aureus-induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

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S. aureusα-toxin is required for induction of apoptosis. (A) Effect of purified α-toxin. Jurkat cells were preincubated for 30 min with various dilutions of a sheep anti–α-toxin antiserum and then incubated with the indicated concentrations of the commercially available preparation of α-toxin. (B and C) Anti–α-toxin neutralizes the proapoptotic activity of S. aureus supernatants. Jurkat cells were preincubated with various dilutions of a sheep anti–α-toxin antiserum, a rabbit anti–α-toxin antiserum, or normal rabbit serum (NRS) for 30 min. Subsequently, sterile-filtered supernatants of RN6390 (B) or Wood 46 (C) were added at the indicated dilutions, and formation of hypodiploid nuclei was assessed. (D) α-Toxin–deficient S. aureus does not induce apoptosis. Jurkat cells were incubated with various concentrations of supernatants of the α-toxin–producing strain DU5883 or its α-toxin–deficient counterpart DU1090. (E) Coomassie-stained SDS-PAGE of a highly purified (lane 2) and the commercially available (lane 3) α-toxin preparation. The molecular sizes of the protein marker used in lane 1 are indicated on the left. (F) Jurkat cells were incubated with the indicated concentrations of the two α-toxin preparations, and cell death was assessed after 24 h.
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fig2: S. aureusα-toxin is required for induction of apoptosis. (A) Effect of purified α-toxin. Jurkat cells were preincubated for 30 min with various dilutions of a sheep anti–α-toxin antiserum and then incubated with the indicated concentrations of the commercially available preparation of α-toxin. (B and C) Anti–α-toxin neutralizes the proapoptotic activity of S. aureus supernatants. Jurkat cells were preincubated with various dilutions of a sheep anti–α-toxin antiserum, a rabbit anti–α-toxin antiserum, or normal rabbit serum (NRS) for 30 min. Subsequently, sterile-filtered supernatants of RN6390 (B) or Wood 46 (C) were added at the indicated dilutions, and formation of hypodiploid nuclei was assessed. (D) α-Toxin–deficient S. aureus does not induce apoptosis. Jurkat cells were incubated with various concentrations of supernatants of the α-toxin–producing strain DU5883 or its α-toxin–deficient counterpart DU1090. (E) Coomassie-stained SDS-PAGE of a highly purified (lane 2) and the commercially available (lane 3) α-toxin preparation. The molecular sizes of the protein marker used in lane 1 are indicated on the left. (F) Jurkat cells were incubated with the indicated concentrations of the two α-toxin preparations, and cell death was assessed after 24 h.

Mentions: We next investigated the nature of the soluble factor in S. aureus culture supernatants, which was responsible for cell death induction. Interestingly, in Coomassie-stained SDS-polyacrylamide gels, we observed a remarkable correlation between the expression levels of α-toxin, the major cytolysin of S. aureus, and the cytotoxic potential of the various S. aureus strains (unpublished data). S. aureus α-toxin has been reported to damage cells by the generation of pores in the plasma membrane (Jonas et al., 1994); however, its precise role in apoptotic pathways has not been established yet. To analyse the cytotoxic potential of this molecule in more detail, Jurkat T cells were incubated with various concentrations of a commercially available α-toxin in the absence or presence of an α-toxin neutralizing antibody. As shown in Fig. 2 A, ∼67% of Jurkat cells were apoptotic after treatment with α-toxin concentrations ranging from 0.1 to 10 μg/ml. The addition of the α-toxin neutralizing antibody resulted in a dose-dependent inhibition of cell death achieved with 0.1 or 1 μg/ml α-toxin. In contrast, the antibody could not inhibit cell death when 10 μg/ml α-toxin were used (Fig. 2 A) or when apoptosis was induced with anti-CD95 (unpublished data), demonstrating the specificity of the α-toxin antibody. More interestingly, when Jurkat cells were treated with supernatants of the moderately and highly cytotoxic S. aureus strains RN6390 (Fig. 2 B) and Wood 46 (Fig. 2 C), respectively, apoptosis could be inhibited completely with two different neutralizing α-toxin antibodies. Normal rabbit serum that was used as a control had no effect. To further verify this finding, we incubated Jurkat cells with supernatants of the α-toxin–producing but FnBP-deficient and thus noninvasive S. aureus strain DU5883 and DU1090, an α-toxin knock-out mutant. Only the supernatant of the α-toxin–producing strain DU5883 was able to induce the dose-dependent death of Jurkat cells, whereas the supernatant of the α-toxin knock-out strain DU1090 had no effect (Fig. 2 D).


alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling.

Bantel H, Sinha B, Domschke W, Peters G, Schulze-Osthoff K, Jänicke RU - J. Cell Biol. (2001)

S. aureusα-toxin is required for induction of apoptosis. (A) Effect of purified α-toxin. Jurkat cells were preincubated for 30 min with various dilutions of a sheep anti–α-toxin antiserum and then incubated with the indicated concentrations of the commercially available preparation of α-toxin. (B and C) Anti–α-toxin neutralizes the proapoptotic activity of S. aureus supernatants. Jurkat cells were preincubated with various dilutions of a sheep anti–α-toxin antiserum, a rabbit anti–α-toxin antiserum, or normal rabbit serum (NRS) for 30 min. Subsequently, sterile-filtered supernatants of RN6390 (B) or Wood 46 (C) were added at the indicated dilutions, and formation of hypodiploid nuclei was assessed. (D) α-Toxin–deficient S. aureus does not induce apoptosis. Jurkat cells were incubated with various concentrations of supernatants of the α-toxin–producing strain DU5883 or its α-toxin–deficient counterpart DU1090. (E) Coomassie-stained SDS-PAGE of a highly purified (lane 2) and the commercially available (lane 3) α-toxin preparation. The molecular sizes of the protein marker used in lane 1 are indicated on the left. (F) Jurkat cells were incubated with the indicated concentrations of the two α-toxin preparations, and cell death was assessed after 24 h.
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Related In: Results  -  Collection

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fig2: S. aureusα-toxin is required for induction of apoptosis. (A) Effect of purified α-toxin. Jurkat cells were preincubated for 30 min with various dilutions of a sheep anti–α-toxin antiserum and then incubated with the indicated concentrations of the commercially available preparation of α-toxin. (B and C) Anti–α-toxin neutralizes the proapoptotic activity of S. aureus supernatants. Jurkat cells were preincubated with various dilutions of a sheep anti–α-toxin antiserum, a rabbit anti–α-toxin antiserum, or normal rabbit serum (NRS) for 30 min. Subsequently, sterile-filtered supernatants of RN6390 (B) or Wood 46 (C) were added at the indicated dilutions, and formation of hypodiploid nuclei was assessed. (D) α-Toxin–deficient S. aureus does not induce apoptosis. Jurkat cells were incubated with various concentrations of supernatants of the α-toxin–producing strain DU5883 or its α-toxin–deficient counterpart DU1090. (E) Coomassie-stained SDS-PAGE of a highly purified (lane 2) and the commercially available (lane 3) α-toxin preparation. The molecular sizes of the protein marker used in lane 1 are indicated on the left. (F) Jurkat cells were incubated with the indicated concentrations of the two α-toxin preparations, and cell death was assessed after 24 h.
Mentions: We next investigated the nature of the soluble factor in S. aureus culture supernatants, which was responsible for cell death induction. Interestingly, in Coomassie-stained SDS-polyacrylamide gels, we observed a remarkable correlation between the expression levels of α-toxin, the major cytolysin of S. aureus, and the cytotoxic potential of the various S. aureus strains (unpublished data). S. aureus α-toxin has been reported to damage cells by the generation of pores in the plasma membrane (Jonas et al., 1994); however, its precise role in apoptotic pathways has not been established yet. To analyse the cytotoxic potential of this molecule in more detail, Jurkat T cells were incubated with various concentrations of a commercially available α-toxin in the absence or presence of an α-toxin neutralizing antibody. As shown in Fig. 2 A, ∼67% of Jurkat cells were apoptotic after treatment with α-toxin concentrations ranging from 0.1 to 10 μg/ml. The addition of the α-toxin neutralizing antibody resulted in a dose-dependent inhibition of cell death achieved with 0.1 or 1 μg/ml α-toxin. In contrast, the antibody could not inhibit cell death when 10 μg/ml α-toxin were used (Fig. 2 A) or when apoptosis was induced with anti-CD95 (unpublished data), demonstrating the specificity of the α-toxin antibody. More interestingly, when Jurkat cells were treated with supernatants of the moderately and highly cytotoxic S. aureus strains RN6390 (Fig. 2 B) and Wood 46 (Fig. 2 C), respectively, apoptosis could be inhibited completely with two different neutralizing α-toxin antibodies. Normal rabbit serum that was used as a control had no effect. To further verify this finding, we incubated Jurkat cells with supernatants of the α-toxin–producing but FnBP-deficient and thus noninvasive S. aureus strain DU5883 and DU1090, an α-toxin knock-out mutant. Only the supernatant of the α-toxin–producing strain DU5883 was able to induce the dose-dependent death of Jurkat cells, whereas the supernatant of the α-toxin knock-out strain DU1090 had no effect (Fig. 2 D).

Bottom Line: Furthermore, alpha-toxin-induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2.Together with our finding that alpha-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus alpha-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors.Hence, our findings clearly define a signaling pathway used in S. aureus-induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Cell Biology, University of Münster, 48149 Münster, Germany.

ABSTRACT
Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and DNA fragmentation were induced not only when Jurkat T cells were infected with intact bacteria, but also after treatment with supernatants of various S. aureus strains. We also demonstrate that S. aureus-induced cell death and caspase activation were mediated by alpha-toxin, a major cytotoxin of S. aureus, since both events were abrogated by two different anti-alpha-toxin antibodies and could not be induced with supernatants of an alpha-toxin-deficient S. aureus strain. Furthermore, alpha-toxin-induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2. Together with our finding that alpha-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus alpha-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors. Hence, our findings clearly define a signaling pathway used in S. aureus-induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

Show MeSH
Related in: MedlinePlus