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Infection and Immunity, December 1998, p. 5980-5987, Vol. 66, No. 12
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Actinobacillus actinomycetemcomitans Toxin Induces Both Cell Cycle Arrest in the G2/M Phase and Apoptosis

Masahiro Ohguchi,1,2 Akira Ishisaki,1 Nobuo Okahashi,1 Masanori Koide,1,2 Takeyoshi Koseki,1 Kenji Yamato,1,3 Toshihide Noguchi,2 and Tatsuji Nishihara1,*

Department of Oral Science, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640,1 Department of Periodontology, School of Dentistry, Aichi Gakuin University, Chikusa-ku, Nagoya 464-8651,2 and Department of Molecular Cellular Oncology/Microbiology, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo 113-8549,3 Japan

Received 9 June 1998/Returned for modification 29 July 1998/Accepted 3 September 1998

We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue.

* Corresponding author. Mailing address: Department of Oral Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111, ext. 2220. Fax: 81-3-5285-1172. E-mail: tatsujin{at}nih.go.jp.

Infection and Immunity, December 1998, p. 5980-5987, Vol. 66, No. 12
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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