A study on the function of cell-cycle specific inhibitor, CDT

伴放线放线杆菌（Actinobacillusactinomycetemcomitans，A. a）的CDT由CDTA、B和C组成，编码cdtA、cdtB和cdtC基因，串联定位于染色体cdt基因座上。免疫沉淀法研究表明CDT形成复合物。对纯化的CDT进行N-末端测序，结果表明CDTA的36-43个氨基酸可能经过脂质修饰后在N-末端进一步加工。蔗糖密度梯度电泳显示CDT复合物定位于外膜。CDT诱导的T细胞系死亡伴随着细胞膜构象改变、核内DNA断裂和caspase活性增加等凋亡生化特征。caspase-2和caspase-7抑制剂对CDT诱导的Jurkat细胞凋亡有明显的抑制作用，表明CDT具有通过激活caspase-2和caspase-7诱导人T细胞凋亡的能力。在45株A. a临床分离株中，所有菌株的细胞裂解液和培养上清中均检测到CDT活性，但培养上清中的毒素滴度在菌株间差异较大。PCR实验表明，在40株菌中存在Y 4型CDT序列，表明CDT生产在A. A菌株中是普遍存在的。

CDT from Actinobacillus actinomycetemcomitans (A.a) is composed of CDTA, B, and C encoded as cdtA, cdtB and cdtC genes tandemly-located on the chromosomal cdt locus. Immunoprecipitation study indicates that CDT forms complex. N-terminal sequencing of purified CDT revealed that 36-43 amino acids of CDTA are further processed at N-terminus probably after lipidmodification of CDTA. Sucrose density gradient ultracentrifuge showed the localization of CDT complex at the outer membrane. Taken together, we propose the hypothetical pathway of CDT secretion from periplasmic space to culture supernatant.CDT-induced death in T cell lines was accompanied with the biochemical features of apoptosis including membrane conformational change, intranucleosomal DNA cleavage, and increase of caspase activity in the cells. Inhibitors for caspase-2 and -7 showed significant inhibitory effect on CDT-induced apoptosis of Jurkat cells suggesting that CDT possesses an ability to induce human T cell apoptosis through activation of caspase-2 and -7.Among 45 A.a clinical isolates, CDT activity was found in cell lysate and culture supernatant of all tested strains, but the titer of the toxin in culture supernatant considerably varied among strains. PCR experiments indicated the presence of Y4-type cdt sequences in forty strains, suggesting that CDT production is prevalent in A.a strains.