Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin
Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin
复制标题
DOI：
10.1111/cmi.13380
复制
发表时间：
2021-08-11
影响因子：
3.4
通讯作者：
Teter，Ken
中科院分区：
文献类型：
--
作者：
Robb Huhn，G.;Torres-Mangual，Naly;Teter，Ken
许多革兰氏阴性菌产生一种细胞致死性膨胀毒素(CDT)，其具有两个细胞结合亚基(CDtA+CDtC)和一个催化的CDtB亚基。CDT黏附于靶细胞的质膜后，通过逆行转运至内质网。然后，CDtB进入细胞核，在那里产生DNA断裂，导致细胞周期停滞和细胞凋亡或衰老。CDtA将CDT全毒素锚定在质膜上，并被认为在CDtB/CdtC异源二聚体内吞后仍留在细胞表面。在这里，我们重新检查了潜在的内吞作用和胞内转运的CDT的嗜血杆菌。我们用基于细胞的酶联免疫吸附试验(CELISA)记录了全毒素相关的CDtA的内吞作用，并在CDT与细胞表面结合10分钟后通过共聚焦显微镜观察到它在早期内吞体内的存在。Western印迹分析证明内化的CDtA迅速降解。大部分内化的CdtB和CdtC也被降解。CDT内化和周转的速度很快，这可以解释为什么以前的研究没有检测到CDtA内吞作用，这表明只有一小部分细胞相关的CDtB到达细胞核。我们的工作证明了CDT被内化为一个完整的全毒素，并确定内体是CDtA从CDtB/CDTC解离的位置。在CDT的内吞过程中，CDtA被认为留在细胞表面。基于细胞的ELISA法记录了CDTA的快速内吞作用。通过共聚焦显微镜观察到早期内体中的CDtA。细胞内的CDtA迅速降解，以及大部分CDtB和CDTC。
Many Gram‐negative pathogens produce a cytolethal distending toxin (CDT) with two cell‐binding subunits (CdtA + CdtC) and a catalytic CdtB subunit. After adhesion to the plasma membrane of a target cell, CDT moves by retrograde transport to endoplasmic reticulum. CdtB then enters the nucleus where it generates DNA breaks that lead to cell cycle arrest and apoptosis or senescence. CdtA anchors the CDT holotoxin to the plasma membrane and is thought to remain on the cell surface after endocytosis of the CdtB/CdtC heterodimer. Here, we re‐examined the potential endocytosis and intracellular transport of CdtA from theHaemophilus ducreyiCDT. We recorded the endocytosis of holotoxin‐associated CdtA with a cell‐based enzyme‐linked immunoabsorbent assay (CELISA) and visualised its presence in the early endosomes by confocal microscopy 10 min after CDT binding to the cell surface. Western blot analysis documented the rapid degradation of internalised CdtA. Most of internalised CdtB and CdtC were degraded as well. The rapid rate of CDT internalisation and turnover, which could explain why CdtA endocytosis was not detected in previous studies, suggests only a minor pool of cell‐associated CdtB reaches the nucleus. Our work demonstrates that CDT is internalised as an intact holotoxin and identifies the endosomes as the site of CdtA dissociation from CdtB/CdtC.Take AwaysDuring the endocytosis of CDT, CdtA is thought to remain at the cell surface.A cell‐based ELISA documented the rapid endocytosis of CdtA.CdtA was visualised in the early endosomes by confocal microscopy.Intracellular CdtA was rapidly degraded, along with most of CdtB and CdtC.