The fate of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin.

Abstract

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi, the causative agent of genital cancroid. CDT is a heterotrimeric toxin consisting of a cell-binding domain (CdtA + CdtC) and a catalytic domain (CdtB) that has DNase activity. After binding to the host plasma membrane, CDT undergoes endocytosis and travels to the endoplasmic reticulum (ER). Only then does CdtB move into the nucleus, causing DNA damage that induces cell-cycle arrest and apoptosis. The previous CDT trafficking model suggested that CdtA remains on the plasma membrane while the CdtB/CdtC heterodimer is transported inside the cell. This model is based on experiments that were unable to detect CdtA inside the host cell. Here, we examine this model and demonstrate that CDT is internalized as an intact holotoxin. Furthermore, the acidification of the endosomes induces CdtA release from the CdtB/CdtC heterodimer. Using a cell-based ELISA, we report that CdtA facilitates CDT binding to the plasma membrane and demonstrate that nearly the entire pool of surface-bound toxin is internalized from the plasma membrane within 20 minutes. As determined by Western blot, all of internalized CdtA and most of internalized CdtB and CdtC are rapidly degraded in the lysosomes. Acidic conditions found in the early endosomes (pH 6.0-6.3) led to a loss of protein structure in CdtA and its release from the CDT holotoxin as determined by circular dichroism and surface plasmon resonance, respectively. These results demonstrate that CDT is internalized as an intact holotoxin, with the acidic environment of endosomes triggering the separation of CdtA from the CdtB/CdtC heterodimer.