Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin

Abstract

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. TAKE AWAYS: During 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.