Molecular Analysis of the Cytolethal Distending Toxin

细胞致死膨胀毒素（CDT）是一种由越来越多的不相关的细菌病原体产生的强毒性细菌毒素，包括分散分离的大肠杆菌和志贺氏杆菌、弯曲杆菌、杜氏嗜血杆菌、放线菌和肠致病性幽门螺杆菌。最初，CDT的特点是能够诱导大量细胞膨胀和细胞死亡。CDT处理的细胞在G2中由于细胞周期的中断而经历不可逆的细胞分裂阻滞。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相似。CDT是cdtA、cdtB和cdtC三个基因的产物，它们编码的蛋白质分子量分别为30kda、32kda和20kda。遗传和生化证据表明，这三种多肽都是细胞中毒所必需的。我们最近报道了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中的dna相关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Mg2plus依赖性dna酶活性。我们也提供证据表明CDT损伤染色体DNA，随后激活DNA损伤检查点级联的元素。虽然单独添加到细胞中没有毒性，但将CdtB引入细胞会导致整个CDT活性谱。因此，我们假设CdtB介导CDT的细胞致死作用，而CdtA和CdtC是CdtB细胞结合和/或转位所必需的。在这个应用程序中，我们建议：1)确定三种CDT多肽在细胞结合和CdtB进入中的作用，2)确定CdtB通过细胞运输并转运到细胞核（CdtB作用的明显部位）的机制，以及3)确定导致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.