Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt): evidence that the holotoxin is composed of three subunits: CdtA, CdtB, and CdtC.
We have shown the Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene, which is homologous to a family of Cdts expressed by several Gram-negative bacteria. We now report that the capacity for CdtB to induce G(2) arrest in Jurkat cells is greater in the presence of the other Cdt peptides: CdtA and CdtC. Plasmids containing the cdt operon were constructed and expressed in Escherichia coli; each plasmid contained a modified cdt gene that expressed a Cdt peptide containing a C-terminal His tag. All three Cdt peptides copurified with the His-tagged Cdt peptide. Each of the peptides associated with the complex was truncated; N-terminal amino acid analysis of CdtB and CdtC indicated that the truncation corresponds to cleavage of a previously described signal sequence. CdtA was present in two forms in crude extracts, 25 and 18 kDa; only the 18-kDa fragment copurified with the Cdt complexes. Cdt complexes were also immunoprecipitated from A. actinomycetemcomitans extracts using anti-CdtC mAb. Exposure of Jurkat cells to 40 pg resulted in >50% accumulation of G(2) cells. CdtB and CdtC were detected by immunofluorescence on the cell surface after 2-h exposure to the holotoxin. CdtA was not detected by immunofluorescence, but all three peptides were associated with Jurkat cells when analyzed by Western blot. These studies suggest that the active Cdt holotoxin is a heterotrimer composed of truncated CdtA, CdtB, and CdtC, and all three peptides appear to associate with lymphocytes.
Shenker
Bruce J
BJ
Department of Pathology, University of Pennsylvania School of Dental Medicine, 240 South 40th Street, Philadelphia, PA 19104-6030, USA. shenker@pobox.dental.upenn.edu.
Besack
Dave
D
McKay
Terry
T
Pankoski
Lisa
L
Zekavat
Ali
A
Demuth
Donald R
DR
D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001483:Base Sequence; D016195:G2 Phase / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D019169:Jurkat Cells; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D011233:Precipitin Tests; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D013601:T-Lymphocytes / Q000187:drug effects / Q000382:microbiology / Q000473:pathology
D001427:Bacterial Toxins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D021122:Protein Subunits; C447141:cytolethal distending toxin, Actinobacillus actinomycetemcomitans