An N-terminal Segment of the Active Component of the Bacterial Genotoxin Cytolethal Distending Toxin B (CDTB) Directs CDTB into the Nucleus

Nishikubo, Shuichi; Ohara, Masaru; Ueno, Yoko; Ikura, Masae; Kurihara, Hidemi; Komatsuzawa, Hitoshi; Oswald, Éric; Sugai, Motoyuki

doi:10.1074/jbc.m305062200

Cited by 82 publications

(90 citation statements)

References 42 publications

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“…Mutants of CdtB lacking DNase activity fail to induce cell cycle arrest (Elwell & Dreyfus, 2000;Lara-Tejero & Galan, 2000;Nesic et al, 2004). The presence of a nuclear localization signal in the A. actinomycetemcomitans CdtB protein also supports the hypothesis that DNA is the molecular target (Nishikubo et al, 2003). This evidence, as well as the proven nuclear localization of CdtB (McSweeney & Dreyfus, 2004), firmly establishes that DNA breakage as a result of CdtB activity, and not some…”

Section: Discussionsupporting

confidence: 62%

Mechanism of internalization of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

et al. 2005

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Cytolethal distending toxin (CDT), which is encoded by three genes, cdtA, cdtB and cdtC, is now recognized to have a growing list of biological actions, including inhibition of cell cycle progression, promotion of apoptosis and stimulation of cytokine secretion. It appears that internalization of CDT is essential, at least for cell cycle blockade. Using purified recombinant CDT proteins from the periodontopathic bacterium Actinobacillus actinomycetemcomitans, the authors investigated which combination of toxin proteins produce cell cycle inhibition and which bound and/or entered into host cells. No evidence was found that CdtB bound to HEp-2 human epithelial cells. In contrast, both CdtA and CdtC bound to these cells. Induction of cell cycle arrest required that cells be exposed to both CdtB and CdtC. Pre-exposure of cells to CdtC for as little as 10 min, followed by removal of the free CdtC and addition of exogenous CdtB, resulted in the inhibition of cell cycle progression, suggesting that CdtB could bind to cell-surface-located CdtC. Using various methods to follow internalization of the CDT proteins it was concluded that CdtC acts to bind CdtB at the cell surface and transports it into the cell as a complex via an endosomal pathway blockable by monensin and brefeldin A.

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Section: Discussionsupporting

confidence: 62%

Mechanism of internalization of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

et al. 2005

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“…3 Nishikubo et al, 2003). An N-terminal NLS has been proposed for AactCdtB, while two C-terminal NLSs have been found in EcolCdtB-II (McSweeney & Dreyfus, 2004;Nishikubo et al, 2003).…”

Section: Cdtmentioning

confidence: 99%

“…3 Nishikubo et al, 2003). An N-terminal NLS has been proposed for AactCdtB, while two C-terminal NLSs have been found in EcolCdtB-II (McSweeney & Dreyfus, 2004;Nishikubo et al, 2003). On the basis of highly conserved N-terminal amino acid sequences corresponding to the putative NLS of AactCdtB among all known bacterial CdtB orthologues, a modular structure consisting of an Nterminal domain responsible for nuclear transport and a C-terminal DNase-like domain capable of exerting DSBs has been proposed (McSweeney & Dreyfus, 2004;Nishikubo et al, 2003).…”

Section: Cdtmentioning

confidence: 99%

“…An N-terminal NLS has been proposed for AactCdtB, while two C-terminal NLSs have been found in EcolCdtB-II (McSweeney & Dreyfus, 2004;Nishikubo et al, 2003). On the basis of highly conserved N-terminal amino acid sequences corresponding to the putative NLS of AactCdtB among all known bacterial CdtB orthologues, a modular structure consisting of an Nterminal domain responsible for nuclear transport and a C-terminal DNase-like domain capable of exerting DSBs has been proposed (McSweeney & Dreyfus, 2004;Nishikubo et al, 2003). Unlike the 'A' subunit of other AB toxins which generally translocates from the ER directly into the cell cytosol by a process of ER-associated degradation (ERAD), translocation of HducCdtB is ERAD-independent with the toxin subunit moving directly from the ER lumen to the nucleoplasm without unfolding (Guerra et al, 2009).…”

Section: Cdtmentioning

confidence: 99%

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Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages

et al. 2011

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Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiationinduced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens. IntroductionJohnson and Lior's seminal observations in the 1980s identified a novel heat-labile toxin in culture filtrates obtained from certain Escherichia coli, Shigella dysenteriae and Campylobacter jejuni strains which caused distinctive and progressive cytoplasmic and nuclear enlargement of cultured mammalian cells, so called cytolethal distending toxin (CDT), and uncovered a novel paradigm amongst bacterial toxins and virulence mechanisms (Johnson & Lior, 1987, 1988a. It was not until many years later that Scott & Kaper (1994) identified the genes encoding CDT in E. coli, which set the stage for fundamental investigations into the ecology, biochemistry and molecular mechanisms of cellular toxicity associated with this novel bacterial toxin (Table 1). While a secreted protein cytotoxin was identified among Haemophilus (Haem.) ducreyi clinical isolates in the early 1990s by Purvén & Lagergård (1992), it was not until the late 1990s that this cytotoxin was conclusively shown to be encoded by a cdt gene cluster with homology to the previously identified E. coli genes (Cope et al., 1997). This discovery extended the range of niches where CDTproducing bacteria are found to include mucocutaneous surfaces of the genital tract in addition to the intestinal tract. At that time, Pérès et al. (1997) first reported that the mechanism of mammalian cell intoxication by E. coli CDT involved arrest of the cell cycle at the G2/M phase. Soon after, these observa...

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“…Interestingly, EcCdtB has smaller clusters (one or two) of Arg/Lys residues than the classical monopartite and bipartite importin-␣ consensus sequences with two to four adjacent Arg/Lys residues (40). It should be noted that in vivo studies of AaCdtB reported an N-terminal region (Asn 48 -Ser 124 ) as the site for an NLS, although no candidate Arg/Lys residues were found in this region (47). A second tandem arginine sequence (Arg 235 -Arg 236 ) in EcCdtB, which is required for cellular entry or cytoplasmic trafficking but is not absolutely required for nuclear localization, is located in the putative EcCdtB-EcCdtC interface (12).…”

Section: Distinct Eccdtb Solution and Crystal States Provide Insightsmentioning

confidence: 99%

Differences in Crystal and Solution Structures of the Cytolethal Distending Toxin B Subunit

Hontz¹,

Villar-Lecumberri²,

Potter

et al. 2006

Journal of Biological Chemistry

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Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15 N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly 233 -Asp 242 ). Residues Leu 186 -Thr 209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined ␣-helix and ␤-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions. Cytolethal distending toxin (CDT)4 is a DNA-damaging bacterial toxin produced by a number of important bacterial pathogens (1, 2). The cytopathic effects associated with CDT intoxication are a direct result of chromosomal DNA damage inflicted by the CdtB subunit, a homolog of eukaryotic type I DNase (3, 4). CdtB-mediated chromosome strand breakage signals the induction of an ATM (ataxia telangiectasia-mutated)-or ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent mitotic checkpoint resulting in the sequestration of Cdc25, a block in the cell cycle at the G 2 /M boundary, cellular distension, and ultimately cell death by endoreduplication or apoptosis (2, 5).The CDT holotoxin is a heterotrimer composed of CdtA, CdtB, and CdtC subunits. Although the catalyst for CDT-mediated apoptosis is ultimately attributed to the DNase activity of CdtB, all three subunits are required for cellular entry of CDT (6, 7). The CdtA and CdtC subunits are lectin-like domains that bind carbohydrate residues on the target cell surface, whereby CDT is subsequently internalized by receptor-mediated endocytosis (8, 9). The Haemophilus ducreyi holotoxin structure (HdCDT) has demonstrated that CDT is an AB type toxin, with the catalytically active A subunit of the AB toxin corresponding to the CdtB subunit (10). Inspection of this structure and of the structurally similar CDT holotoxin from Actinobacillus actinomycetemcomitans (AaCDT) (11) revealed that one face of the CdtA-CdtC dimer binds the CdtB subunit. Mutagenesis studies of HdCDT suggest that another face of the dimer with a grooved ricin B-chain fold mediates cell surface binding (10).Trafficking of CDT to the nucleus is thought to involve retrograde transport through the Golgi and the ultimate translocation of CdtB from the endoplasmic reticulum to the nuclear envelope by an apparent ERAD (endoplasmic reticulum-associated degradation)-independent step (9). CdtB traverse...

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An N-terminal Segment of the Active Component of the Bacterial Genotoxin Cytolethal Distending Toxin B (CDTB) Directs CDTB into the Nucleus

Cited by 82 publications

References 42 publications

Mechanism of internalization of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Mechanism of internalization of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages

Differences in Crystal and Solution Structures of the Cytolethal Distending Toxin B Subunit

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