Objective: Cytolethal distending toxin (CDT) is one of exotoxins produced by Actinobacillus actinomycetemcomitans. CDT causes cell distending and cell cycle arrest, and eventually elicits cell death against KB cells. While various Gram-negative pathogens are known to produce CDTs, any native CDT has not been purified yet. In this study, we established the purification system for the native but not reconstituted A. actinomycetemcomitans CDT. Methods: Purification of the toxin complex is achieved by introduction of a histidine tag into the C-terminal end of CdtB. Results: A purified CDT complex (wtCDT) is composed of heterogeneous CdtA (subunit A), 31 kDa CdtB (subunit B; fused with a histidine tag), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. The heterogeneous CdtA proteins were detected as 24.5 kDa and 18 kDa proteins in nearly a 0.2:0.8 stoichiometry. This is the first evidence for the native holotoxin structure of CDT. To eliminate the heterogeneity of subunit A, we constructed the in-flame deletion mutant in the N-terminal region of CdtA, which overlaps the additional processing region of CdtA. We identified the mutant which produced an active CDT and the toxin complex purified to homogeneity. Conclusion: The purified CDT showed the same property with purified wtCDT but is composed of 18.5 kDa subunit A, 31 kDa subunit B, and 18.5 kDa subunit C in almost a 1:1:1 stoichiometry, indicating the additional processing region of CdtA is dispensable for the toxin.

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