A New Functional Site W115 in CdtA Is Critical for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtAY181ABC holotoxin and the biological function of CDT was not weaken in CdtAY105ABC, CdtAY125ABC, CdtAF109ABC and CdtAS106NBC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtAW115GBC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.

伴放线放线杆菌（Aggregatibacter actinomycetemcomitans）是局限性侵袭性牙周炎的特异性致病菌，可产生细胞致死膨胀毒素（cytolethal distending toxin, CDT），该毒素能将真核细胞不可逆地阻滞于细胞周期的G0/G1期或G2/M期。尽管结构研究表明CdtA的芳香簇区域（aromatic patch region）在其生物学活性中发挥重要作用，但目前尚未明确CdtA的功能性结合位点。本研究采用定点诱变（site-specific mutagenesis）策略构建cdtA基因的点突变体，以探究单个氨基酸残基对受体结合以及全毒素（holotoxin）生物学活性的贡献。实验结果显示，CdtAY181ABC全毒素的结合能力有所下降；而CdtAY105ABC、CdtAY125ABC、CdtAF109ABC与CdtAS106NBC全毒素的CDT生物学功能并未受到削弱，提示上述位点并非CDT发挥活性的关键位点。但CdtAW115GBC全毒素复合物的结合活性与细胞周期阻滞能力均受到抑制，且经尺寸排阻色谱（size exclusion chromatography）验证，该位点并未影响全毒素的组装过程。因此，W115可能是CdtA结合能力的关键位点。本研究结果表明，CdtA的功能性位点并非仅局限于芳香簇区域。W115作为全新的功能性位点，对受体结合与细胞周期阻滞均发挥关键作用，为通过药理学手段干预CDT活性提供了潜在靶点。