Biogenesis of Actinobacillus actinomycetemcomitans Cdt holotoxin

Ueno Yoko

Ohara Masaru

Kawamoto Toru

Fujiwara Tamaki

Komatsuzawa Hitoshi

Oswald Eric

Sugai Motoyuki

Infection and Immunity

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 E.coli carrying pTK3022, we show the sixteenth residue, cysteine, of CdtA bound [3H]palmitate or [3H]glycerol. Further post translational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblot show the lipid modified CdtA is present in the outer membrane. Immuno-precipitation and the pull-down assay of the Cdt complex from E. coli carrying a plasmid containing CdtABC demonstrated the Cdt complex in the periplasm is composed of CdtA, CdtB, and CdtC, and that the Cdt complex in culture supernatant is a N-terminally truncated (36-43 amino acids) form of CdtA (CdtA'), CdtB, and CdtC. This suggests Cdt is present as a complex both in the periplasm and the supernatant where CdtA undergoes post-translation processing to CdtA' in the process of biogenesis and secretion of Cdt holotoxin into the culture supernatant. Site-directed mutagenesis of the sixteenth 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 Cdt forms a complex inside the periplasm for lipid modification where post-translational processing of CdtA plays an important role for the efficient production of Cdt holotoxin into the culture supernatant.

Cdt

lipoprotein

Actinobacillus actinomycetemcomitan

Medical sciences [ 490 ]

eng

American Society for Microbiology

Copyright (c) 2006 American Society for Microbiology.

[ISSN] 0019-9567

[DOI] 10.1128/IAI.00739-05

[NCID] AA00673732

[PMID] 16714579

[DOI] http://dx.doi.org/10.1128/IAI.00739-05 isVersionOf