Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that A[Delta]₁₉₋₄₇, with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA. The wild-type CDT (wtCDT) and the mutant CDT (A[subscript [Delta]]₁₉₋₄₇CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified A[subscript [Delta]]₁₉₋₄₇CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in A[subscript [Delta]]₁₉₋₄₇CDT. Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer. In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity. Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.