Molecular Analysis of the Cytolethal Distending Toxin

细胞致死性膨胀毒素（CDT）是一种强效的细菌毒素，由越来越多的无关细菌病原体产生，包括零星的大肠杆菌分离株。大肠杆菌和志贺氏菌分离株，弯曲杆菌属，杜克雷嗜血杆菌、伴放线放线杆菌和肠致病性螺杆菌。最初，CDT的特征在于其诱导大量细胞膨胀和细胞死亡的能力。 用CDT处理的细胞经历由G2中细胞周期的破坏引起的细胞分裂的不可逆阻断。 导致CDT介导的生长停滞的特定事件与诱导有丝分裂DNA损伤检查点后的事件平行。 CDT是命名为cdtA、cdtB和cdtC的三个基因的产物，其编码分子量为30、32和20 kDa的蛋白质。 遗传和生物化学证据表明，所有三种多肽都是细胞中毒所必需的。 我们最近报道CdtB与哺乳动物I型DNA酶具有重要的位置特异性序列相关性。 突变分析表明CdtB中与DNA酶相关的活性位点残基是生物活性所必需的。初步研究表明，纯化的CdtB具有Mg ~（2+）依赖的DNA酶活性。 我们还提出的证据表明，CDT损伤染色体DNA，随后激活的DNA损伤检查点级联的元素。 虽然当单独添加到细胞中时没有毒性，但将CdtB引入细胞中导致整个CDT活性谱。 因此，我们假设CdtB介导CDT的细胞致死效应，而CdtA和CdtC是CdtB的细胞结合和/或易位所需的。 在本申请中，我们提出：1）定义三种CDT多肽中的每一种在细胞结合和CdtB进入中的作用，2）确定CdtB通过细胞运输并易位到细胞核（CdtB作用的表观位点）中的机制，和3）定义导致CDT介导的生长停滞和死亡的一系列事件。 这些目标的完成将为这种强效细菌毒素的新作用提供新的见解。

The cytolethal distending toxin (CDT) is a potent bacterial toxin produced by a growing list of unrelated bacterial pathogens including scattered isolates of E. coli and Shigella isolates, Campylobacter spp., Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enteropathogenic Helicobacter spp. Initially, CDT was characterized by its capacity to induce massive cellular distension and cell death. Cells treated with CDT undergo an irreversible block in cell division caused by disruption of the cell cycle in G2. The specific events leading to CDT-mediated growth arrest parallel those following induction of the mitotic DNA damage checkpoint. CDT is the product of three genes designated cdtA, cdtB, and cdtC, which encode proteins with molecular masses of 30, 32, and 20 kDa. Genetic and biochemical evidence indicate that all three polypeptides are required for cellular intoxication. We recently reported that CdtB bears sidnificance position-specific sequence relatedness to mammalian type I DNase. Mutational analysis indicates that the DNase-related active site residues in CdtB are required for biological activity. In preliminary studies show that purified CdtB possesses Mg2plus- dependent DNase activity. We also present evidence indicating that CDT damages chromosomal DNA followed by activation of elements of the DNA damage checkpoint cascade. Although not toxic when added alone to cells, introduction of CdtB into cells results in the entire spectrum of CDT activities. We therefore hypothesize that CdtB mediates the cytolethal effects of CDT while CdtA and CdtC are required for cell binding and/or translocation of CdtB. In this application we propose to: 1) define the role of each of the three CDT polypeptides in cell binding and CdtB entry, 2) determine the mechanism by which CdtB traffics through the cell and translocates into nucleus (the apparent site of CdtB action, and 3) define the series of events leading to CDT-mediated growth arrest and death. Completion of these aims will provide new insights into the novel action of this potent bacterial toxin.