Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans
Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans
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DOI：
10.1111/j.1365-2958.2005.04905.x
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发表时间：
2005-12-01
影响因子：
3.6
通讯作者：
DiRienzo, JM
中科院分区：
文献类型：
--
作者：
Cao, L;Volgina, A;DiRienzo, JM
CDT是一类革兰氏阴性细菌毒素，通常使真核细胞停留在细胞周期的G(0)/G(1)或G2/M期。该毒素是由cdtA、cdtB和cdtC基因产物组成的杂三聚体。尽管在培养和酶联免疫吸附试验(CELISA)中已证明CDtA蛋白亚基与细胞结合，但其与CDtB和CDtC相互作用的确切机制尚不清楚。在这项研究中，我们采用随机突变策略来构建cdtA的点突变文库，以评估单个氨基酸对结合活性的贡献以及亚基形成生物活性全毒素的能力。在7个cdtA突变体中，单一独特的氨基酸替换导致纯化的重组蛋白与中国仓鼠卵巢细胞的结合减少，并与含有岩藻糖的糖蛋白甲状腺球蛋白失去结合。这些突变聚集在cdtA基因的5‘端和3’端，导致位于芳香斑片区和cdtA同源物保守区之外的氨基酸替换。S165N(MutA81)、T41a(MutA121)和C178W(MutA221)上的三个氨基酸替换导致基因产物形成全毒素复合体，其增殖抑制作用降低60%(MutA81)或丧失60%(mutA121、mutA221)。当在体外测试这些突变的全毒素诱导细胞周期停滞和将超螺旋DNA转化为松弛和线性形式的能力时，也观察到了类似的模式。突变A81和突变A221的突变破坏了全毒素的形成。氨基酸取代的位置被绘制在杜氏嗜血杆菌CDT晶体结构中，为了解结构和功能提供了一些信息。
The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G(0)/G(1) or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5'- and 3'-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.