Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans.
Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.
Saiki
K
K
Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Japan. keisaiki@tokyo.ndu.ac.jp
Konishi
K
K
Gomi
T
T
Nishihara
T
T
Yoshikawa
M
M
D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D006131:Growth Inhibitors / Q000302:isolation & purification; D007166:Immunosuppressive Agents / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis
D001427:Bacterial Toxins; D006131:Growth Inhibitors; D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; C067044:cytolethal distending toxin