Biogenesis of the Actinobacillus actinomycetemcomitans cytolethal distending toxin holotoxin.
The cell cycle G2/M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA, cdtB, and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called "lipobox", at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [3H]palmitate or [)H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA'), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA' in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant.
Ueno
Yoko
Y
Department of Bacteriology, Hiroshima University Graduate School of Biomedicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.
Ohara
Masaru
M
Kawamoto
Toru
T
Fujiwara
Tamaki
T
Komatsuzawa
Hitoshi
H
Oswald
Eric
E
Sugai
Motoyuki
M
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D006027:Glycosides / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014315:Triterpenes
D001427:Bacterial Toxins; D006027:Glycosides; D008074:Lipoproteins; D014315:Triterpenes; C447141:cytolethal distending toxin, Actinobacillus actinomycetemcomitans; D019308:Palmitic Acid; C001883:stichoposide