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Infection and Immunity, December 2004, p. 7055-7062, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7055-7062.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Streptococcal Pyrogenic Exotoxin B-Induced Apoptosis in A549 Cells Is Mediated by a Receptor- and Mitochondrion-Dependent Pathway

Wan-Hua Tsai,^1,2 Chia-Wen Chang,^1,2 Woei-Jer Chuang,^1,2 Yee-Shin Lin,^1,3 Jiunn-Jong Wu,^1,4 Ching-Chuan Liu,⁵ Wen-Tsan Chang,^1,2 and Ming T. Lin^1,2*

Institutes of Basic Medical Sciences,¹ Biochemistry,² Microbiology and Immunology,³ Medical Technology,⁴ Department of Pediatrics, National Cheng Kung University Medical College, Tainan, Taiwan⁵

Received 19 December 2003/ Returned for modification 6 May 2004/ Accepted 19 August 2004

It has been shown that streptococcal pyrogenic exotoxin B (SPE B) can induce cells to undergo apoptosis. The present study is to dissect the role of SPE B protease and SPE B protein in the apoptotic process of A549 cells and to elucidate the SPE B-induced apoptotic pathway. Recombinant SPE B (rSPE B) and C192S, a mutant of SPE B without protease activity, were expressed in Escherichia coli and purified by using an affinity column. The apoptosis of A549 cells was assayed by propidium iodide staining, followed by flow cytometry analysis. Our results showed that SPE B induced apoptosis in a dose-dependent manner, whereas C192S did not. When cells were pretreated with rSPE B (2 µg/ml) for as briefly as 5 min and then incubated with C192S of 28 kDa, an apoptosis that is proportional to the period of pretreatment was observed but not with C192S of 42 kDa. These results suggest that the extracellular protease activity of rSPE B is required for the initiation of apoptosis and that the size of SPE B is important for an effective induction of apoptosis. The time course analysis revealed that molecules activated in apoptosis were in the following order: caspase-8 (1.5 h), t-Bid (2.5 h), Bax (3 h), cytochrome c release (6 h), caspase-9 (7 h), and caspase-3 (8 h). The overexpression of Bcl-2 inhibited depolarization of mitochondrial membrane, cytochrome c release, and apoptosis. The results of the present study suggest that SPE B-induced apoptosis is mediated through a receptor-like mechanism and a mitochondrion-dependent pathway.

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* Corresponding author. Mailing address: Department of Biochemistry, National Cheng Kung University Medical College, 1 Da Shuei Rd., Tainan 701, Taiwan. Phone: 886-6-235-3535, ext. 5544. Fax: 886-6-274-1694. E-mail: lin1223{at}mail.ncku.edu.tw.

Editor: V. J. DiRita

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Infection and Immunity, December 2004, p. 7055-7062, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7055-7062.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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