University of Central Florida Orlando, Florida, United States
Isabel Silva (University of Central Florida)| Carla Reyes (University of Central Florida)| Ashley Fragoso (University of Central Florida)| G. Robb Huhn (University of Central Florida)| Ken Teter (University of Central Florida)
Haemophilus ducreyi is a Gram-negative bacterium responsible for the sexually transmitted disease chancroid. It releases a heterotrimeric cytolethal distending toxin (CDT) that contains a catalytic subunit (CdtB) which has a DNAse activity and two binding components (CdtA and CdtC) responsible for entry into the cell. Our hypothesis predicts that the endocytosed holotoxin disassembles through two steps, where CdtA is dissociated from CdtB/CdtC subunits in the late endosomes due to the low pH of this compartment. Afterward, CdtC is released in the endoplasmic reticulum. This allows CdtB to enter the nucleus, causing DNA damage and cell cycle arrest.
To study the mechanism of CdtA dissociation, each Cdt subunit was expressed in bacteria transformed with Cdt expression plasmids. Subunit purification was confirmed using SDS-PAGE with Coomassie stain. Afterward, circular dichroism studies tested the structural stabilities of each Cdt subunit at extracellular pH (7.4), early endosome pH (6.3), and late endosome pH (5.2). These structural studies showed that CdtB and CdtC do not undergo significant secondary structure changes at acidic pH. Acid-induces changes were observed with the CdtA subunit, causing its unfolding, aggregation and precipitation at pH 5.2. Additional studies assembled the holotoxin from the individual subunits and introduced it to different pH levels to evaluate the association and dissociation of the subunits using size exclusion chromatography. The results showed that the active holotoxin disassembles at a pH of 5.2, suggesting that the toxin dissociates in the acidified endosomal compartments due to the unfolding of CdtA. Knowledge of the CDT dissociation mechanism could be used to find treatments for chancroid that would prevent toxin function by blocking its disassembly.