title: Deletion and purification studies to elucidate the structure of the Actinobacillus actinomycetemcomitans cytolethal distending toxin.
abstract: 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 ADelta19-47, 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 (ADelta19-47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified ADelta19-47CDT 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 ADelta19-47CDT. 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.
authors.affiliation: Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159, Japan. keisaiki@tky.ndu.ac.jp
authors.forename: Keitarou
authors.initials: K
authors.lastname: Saiki
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Gomi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Konishi
mesh_terms: D000188:Actinobacillus / Q000235:genetics*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics*; D015153:Blotting, Western; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
chemical_list: D001427:Bacterial Toxins; D010455:Peptides; D011994:Recombinant Proteins; C067044:cytolethal distending toxin; D013395:Sucrose; D001216:Asparagine; D011392:Proline