Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays–the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays.细胞致死膨胀毒素（Cytolethal Distending Toxin, CDT）由多种致病菌产生。已知CDT可通过其催化亚基CdtB诱导宿主真核细胞的基因组DNA损伤。CdtB在结构上与脱氧核糖核酸酶I（DNase I）同源，且具有依赖于多个关键残基的核酸酶活性。然而目前已观察到不同CdtB亚基的活性存在差异，且生化数据与细胞实验数据之间存在不一致之处。为更好地表征CdtB在诱导DNA损伤中的作用，我们在天然与变性条件下，分别从大肠杆菌（E. coli）和杜克雷嗜血杆菌（H. ducreyi）中亲和纯化了野生型及突变型CdtB。随后我们通过经典的体外质粒DNA核酸酶活性检测实验，以及两种不同的真核细胞实验对其核酸酶活性进行了比较：第一种实验为将编码CdtB的质粒转染宿主细胞，第二种实验则为直接将纯化的CdtB转染宿主细胞。本研究结果显示，体外核酸酶活性的定量难度较高，而宿主细胞内的CdtB活性则更易于解读，并验证了催化突变体的功能丧失。我们的研究结果强调了在研究细菌基因毒素的作用时开展多实验验证的重要性，同时指出经典体外实验应当辅以细胞水平实验。