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Infection and Immunity, June 2007, p. 2894-2902, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.01639-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Involvement of Caspase-9 in the Inhibition of Necrosis of RAW 264 Cells Infected with Mycobacterium tuberculosis{triangledown}

Ryosuke Uchiyama,1 Ikuo Kawamura,1* Takao Fujimura,2 Michiko Kawanishi,1 Kohsuke Tsuchiya,1 Takanari Tominaga,1 Taijin Kaku,1 Yutaka Fukasawa,1 Shunsuke Sakai,1 Takamasa Nomura,1 and Masao Mitsuyama1

Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan,1 Department of Dermatology, Kitasato University School of Medicine, Sagamihara 228-8555, Japan2

Received 11 October 2006/ Returned for modification 22 November 2006/ Accepted 24 March 2007

In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.

* Corresponding author. Mailing address: Department of Microbiology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4447. Fax: 81-75-753-4446. E-mail: ikuo_kawamura{at}mb.med.kyoto-u.ac.jp

{triangledown} Published ahead of print on 2 April 2007.

Editor: J. L. Flynn

Infection and Immunity, June 2007, p. 2894-2902, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.01639-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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