An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus.
Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.
Nishikubo
Shuichi
S
Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi Minami-ku, Hiroshima 734-8553, Japan.
Ohara
Masaru
M
Ueno
Yoko
Y
Ikura
Masae
M
Kurihara
Hidemi
H
Komatsuzawa
Hitoshi
H
Oswald
Eric
E
Sugai
Motoyuki
M
D021581:Active Transport, Cell Nucleus; D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002875:Chromosomes / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D016195:G2 Phase; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001427:Bacterial Toxins; D002843:Chromatin; D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C067044:cytolethal distending toxin; D049452:Green Fluorescent Proteins; C038753:leptomycin B