Nishikubo Shuichi, Ohara Masaru, Ueno Yoko, Ikura Masae, Kurihara Hidemi, Komatsuzawa Hitoshi, Oswald Eric, Sugai Motoyuki
Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi Minami-ku, Hiroshima 734-8553, Japan.
J Biol Chem. 2003 Dec 12;278(50):50671-81. doi: 10.1074/jbc.M305062200. Epub 2003 Aug 28.
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.
伴放线放线杆菌产生的细胞致死性膨胀毒素（CDT）是牙周疾病发病机制中一种假定的毒力因子。它是细胞周期特异性抑制剂，作用于G2/M期转换。CDT全毒素的亚基之一CDTB，在进入靶细胞后具有基因毒性作用，由此导致的染色体损伤会引发检查点磷酸化级联反应。将CDTB显微注射到细胞质中后，其定位于细胞核并诱导染色质塌陷。为了研究CDTB核转运的分子机制，我们使用了CDTB-绿色荧光蛋白（GFP）融合蛋白的瞬时表达和显微注射技术。显微注射后，带有组氨酸标签的CDTB-GFP在3 - 4小时内进入细胞核。雷帕霉素B并没有提高融合蛋白的进入速度，这表明融合蛋白进入速度相对较慢并非由于该蛋白依赖CRM1的核输出。CDTB-GFP的核定位具有温度依赖性。体外转运实验表明，CDTB的核定位是由主动转运介导的。一系列截短的CDTB-GFP融合蛋白的瞬时表达实验表明，48 - 124位残基构成了CDTB核转运的最小区域。将CDTB核转运相关区域与SV40 T核定位信号进行结构域交换实验表明，CDTB由两个结构域组成，一个是用于核转运的N端结构域和一个C端活性结构域。我们的结果有力地表明，CDTB的核定位是全毒素诱导细胞膨胀和细胞周期阻滞所必需的。这是首次证明一种具有独特核转运结构域的细菌毒素通过主动转运进入动物细胞核。
