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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. aureus induces activation of the initiator caspases 8 and 9. (A) Jurkat cells were incubated with the indicated dilutions of Wood 46 supernatant (SN). After 4 h, cell extracts were prepared and analyzed for caspase activity using the fluorogenic substrates IETD AMC for caspase 8 (▪) and LEHD AMC for caspase 9 (•). The two dashed lines represent IETDase and LEHDase activity in untreated cells. (B) Western blot analysis of the status of caspase-8 (top) and caspase-9 (bottom) in Jurkat cells that were either left untreated (control) or incubated for 4 h with the indicated dilutions of Wood 46 supernatant (SN). The uncleaved proforms and the p43/p41 fragments of caspase-8 and the p37 fragment of caspase-9 are indicated.
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fig6: S. aureus induces activation of the initiator caspases 8 and 9. (A) Jurkat cells were incubated with the indicated dilutions of Wood 46 supernatant (SN). After 4 h, cell extracts were prepared and analyzed for caspase activity using the fluorogenic substrates IETD AMC for caspase 8 (▪) and LEHD AMC for caspase 9 (•). The two dashed lines represent IETDase and LEHDase activity in untreated cells. (B) Western blot analysis of the status of caspase-8 (top) and caspase-9 (bottom) in Jurkat cells that were either left untreated (control) or incubated for 4 h with the indicated dilutions of Wood 46 supernatant (SN). The uncleaved proforms and the p43/p41 fragments of caspase-8 and the p37 fragment of caspase-9 are indicated.

Mentions: Caspase-3 activation can be achieved either by caspase-8, the most proximal caspase in death receptor signaling, or by caspase-9, the initiator caspase of the mitochondrial pathway. To elucidate which of these two pathways is responsible for α-toxin–induced caspase-3 activation, extracts of Wood 46–treated Jurkat cells were analyzed for their capability to cleave the fluorogenic substrates N-acetyl-Ile-Glu-Thr-Asp (IETD)-AMC and N-acetyl-Leu-Glu-His-Asp (LEHD)-AMC, which are indicative of caspase-8 and -9 activity, respectively. In addition, Western blot analyses were performed. Similar to caspase-3 activation (Fig. 3), Wood 46 induced a dose-dependent increase of IETDase and LEHDase activity in Jurkat cells (Fig. 6 A). These enzymatic activities correlated well with the appearance of the p41/p43-processed caspase-8 subunits (Fig. 6 B, top) and the p37 active caspase-9 fragment, respectively (Fig. 6 B, bottom). Wood 46–induced caspase-8 and -9 activation reached a peak when 0.06% of the supernatant was used and declined rapidly with higher concentrations, which is reminiscent of the activation profile of caspase-3 (Fig. 3 C and Fig. 4 A). Similar results were obtained with purified α-toxin (unpublished data), indicating that both caspase-8 and caspase-9 are involved in S. aureus–mediated cytotoxicity.


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. aureus induces activation of the initiator caspases 8 and 9. (A) Jurkat cells were incubated with the indicated dilutions of Wood 46 supernatant (SN). After 4 h, cell extracts were prepared and analyzed for caspase activity using the fluorogenic substrates IETD AMC for caspase 8 (▪) and LEHD AMC for caspase 9 (•). The two dashed lines represent IETDase and LEHDase activity in untreated cells. (B) Western blot analysis of the status of caspase-8 (top) and caspase-9 (bottom) in Jurkat cells that were either left untreated (control) or incubated for 4 h with the indicated dilutions of Wood 46 supernatant (SN). The uncleaved proforms and the p43/p41 fragments of caspase-8 and the p37 fragment of caspase-9 are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198876&req=5

fig6: S. aureus induces activation of the initiator caspases 8 and 9. (A) Jurkat cells were incubated with the indicated dilutions of Wood 46 supernatant (SN). After 4 h, cell extracts were prepared and analyzed for caspase activity using the fluorogenic substrates IETD AMC for caspase 8 (▪) and LEHD AMC for caspase 9 (•). The two dashed lines represent IETDase and LEHDase activity in untreated cells. (B) Western blot analysis of the status of caspase-8 (top) and caspase-9 (bottom) in Jurkat cells that were either left untreated (control) or incubated for 4 h with the indicated dilutions of Wood 46 supernatant (SN). The uncleaved proforms and the p43/p41 fragments of caspase-8 and the p37 fragment of caspase-9 are indicated.
Mentions: Caspase-3 activation can be achieved either by caspase-8, the most proximal caspase in death receptor signaling, or by caspase-9, the initiator caspase of the mitochondrial pathway. To elucidate which of these two pathways is responsible for α-toxin–induced caspase-3 activation, extracts of Wood 46–treated Jurkat cells were analyzed for their capability to cleave the fluorogenic substrates N-acetyl-Ile-Glu-Thr-Asp (IETD)-AMC and N-acetyl-Leu-Glu-His-Asp (LEHD)-AMC, which are indicative of caspase-8 and -9 activity, respectively. In addition, Western blot analyses were performed. Similar to caspase-3 activation (Fig. 3), Wood 46 induced a dose-dependent increase of IETDase and LEHDase activity in Jurkat cells (Fig. 6 A). These enzymatic activities correlated well with the appearance of the p41/p43-processed caspase-8 subunits (Fig. 6 B, top) and the p37 active caspase-9 fragment, respectively (Fig. 6 B, bottom). Wood 46–induced caspase-8 and -9 activation reached a peak when 0.06% of the supernatant was used and declined rapidly with higher concentrations, which is reminiscent of the activation profile of caspase-3 (Fig. 3 C and Fig. 4 A). Similar results were obtained with purified α-toxin (unpublished data), indicating that both caspase-8 and caspase-9 are involved in S. aureus–mediated cytotoxicity.

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