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Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin
Cellular Microbiology ( IF 3.4 ) Pub Date : 2021-07-22 , DOI: 10.1111/cmi.13380
G Robb Huhn 1 , Naly Torres-Mangual 1, 2 , John Clore 1 , Lucia Cilenti 1 , Teresa Frisan 3, 4 , Ken Teter 1
Affiliation  

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 the Haemophilus ducreyi CDT. 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.

中文翻译:

杜克雷嗜血杆菌致细胞膨胀毒素 CdtA 亚基的内吞作用

许多革兰氏阴性病原体产生具有两个细胞结合亚基 (CdtA + CdtC) 和一个催化 CdtB 亚基的细胞致死膨胀毒素 (CDT)。在粘附到靶细胞的质膜后,CDT 通过逆行运输移动到内质网。然后 CdtB 进入细胞核,在那里它产生导致细胞周期停滞和细胞凋亡或衰老的 DNA 断裂。CdtA 将 CDT holotoxin 锚定在质膜上,并被认为在 CdtB/CdtC 异二聚体的内吞作用后保留在细胞表面。在这里,我们重新检查了杜克雷嗜血杆菌中 CdtA 的潜在内吞作用和细胞内转运CDT。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 CdtA 的内吞作用,并在 CDT 与细胞表面结合后 10 分钟通过共聚焦显微镜观察其在早期内体中的存在。蛋白质印迹分析记录了内化 CdtA 的快速降解。大多数内化的 CdtB 和 CdtC 也被降解。CDT 内化和周转的快速速度可以解释为什么在以前的研究中未检测到 CdtA 内吞作用,这表明只有少量细胞相关的 CdtB 到达细胞核。我们的工作表明 CDT 被内化为完整的全毒素,并将内体识别为 CdtA 从 CdtB/CdtC 解离的位点。
更新日期:2021-07-22
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