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Infection and Immunity, August 2005, p. 4846-4852, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4846-4852.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Involvement of Ganglioside GM3 in G₂/M Cell Cycle Arrest of Human Monocytic Cells Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Departments of Preventive Dentistry,¹ Oral Microbiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580,³ Laboratory of Veterinary Immunology, Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka 599-8531, Japan²

Received 15 January 2005/ Returned for modification 21 March 2005/ Accepted 29 March 2005

Actinobacillus actinomycetemcomitans produces a toxin called cytolethal distending toxin (CDT), which causes host cell DNA damage leading to the induction of DNA damage checkpoint pathways. CDT consists of three subunits, CdtA, CdtB, and CdtC. CdtB is the active subunit of CDT and exerts its effect as a nuclease that damages nuclear DNA, triggering cell cycle arrest. In the present study, we confirmed that the only combination of toxin proteins causing cell cycle arrest was that of all three recombinant CDT (rCDT) protein subunits. Furthermore, in order for rCDT to demonstrate toxicity, it was necessary for CdtA and CdtC to access the cell before CdtB. The coexistence of CdtA and CdtC was necessary for these subunits to bind to the cell. Cells treated with the glucosylceramide synthesis inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol showed resistance to the cytotoxicity induced by rCDT. Furthermore, LY-B cells, which are deficient in the biosynthesis of sphingolipid, also showed resistance to the cytotoxicity induced by rCDT. To evaluate the binding of each subunit for glucosylceramides, we performed thin-layer chromatography immunostaining. The results indicated that each subunit reacted with the glycosphingolipids GM1, GM2, GM3, Gb3, and Gb4. The rCDT mixture incubated with liposomes containing GM3 displayed partially reduced toxicity. These results indicate that GM3 can act as a CDT receptor.

Editor: J. T. Barbieri

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