Molecular Analysis of the Cytolethal Distending Toxin

细胞致死性膨胀毒素(CDT)是由越来越多的无关细菌产生的一种有效的细菌毒素，包括散布的大肠杆菌和志贺氏菌、弯曲杆菌、杜热氏嗜血杆菌、伴生放线放线杆菌和肠道致病螺杆菌。最初，CDT的特征是能够诱导大量细胞膨胀和细胞死亡。CDT处理的细胞在细胞分裂过程中经历不可逆转的阻碍，这是由于G2期细胞周期的中断造成的。导致CDT介导的生长停滞的特定事件与诱导有丝分裂DNA损伤检查点之后的事件相似。CDT是三个基因的产物，分别命名为cdtA、cdtB和cdtC，编码分子质量分别为30、32和20 kDa的蛋白质。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酶具有显著的位置特异性序列关系。突变分析表明，CDtB中与DNA酶相关的活性部位残基是生物活性所必需的。初步研究表明，纯化的CdtB具有依赖于镁离子的DNA酶活性。我们还提出了证据表明，CDT损伤染色体DNA，然后激活DNA损伤检查点级联反应的元件。虽然单独添加到细胞中没有毒性，但将CdtB引入细胞会导致CDT活性的整个光谱。因此，我们假设CDtB介导了CDT的细胞毒性效应，而CDtB的细胞结合和/或移位则需要CDtA和CDtC。在这一应用中，我们建议：1)确定三种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通过细胞并移位到细胞核的机制(CDtB作用的表观位置)；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大的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.