Molecular Basis for the Two-Stage Disassembly of Cytolethal Distending Toxin - Cytolethal distending toxin (CDT) is a tripartite genotoxin with a single catalytic subunit (CdtB) and two cell- binding subunits (CdtA + CdtC). This AB-type toxin is produced by numerous Gram-negative pathogens and has been intensively studied because of its possible link to cancer: it damages the DNA of a target cell and could thus cause genomic instability leading to oncogenesis. To reach the host DNA, CDT moves by vesicle carriers from the cell surface through the endosomes and Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi, and CdtB separates from CdtC in the ER. The free CdtB subunit then crosses the ER membrane and enters the nucleus where it acts as a DNase and, possibly, a PIP3 phosphatase. No other AB toxin has a two-stage mechanism to separate the catalytic and cell-binding subunits. CdtB is not active until it dissociates from the two cell-binding components of the toxin, yet the molecular mechanism for disassembly of the CDT holotoxin remains unknown. The goal of this project is to elucidate the molecular details of this essential process in CDT intoxication. We recently reported that, after endocytosis of the intact Haemophilus ducreyi CDT holotoxin, the low pH of the endosomes induces the separation of CdtA from CdtB/CdtC. Preliminary experiments indicate that CdtB dissociates from CdtC in the presence of ATP, which is present in the ER but not the endosomes or Golgi. ATP only binds to the CdtB subunit, but it does not alter the structure of CdtB. In addition, ATP does not affect the stability of the tripartite CDT holotoxin. The endosome-localized dissociation of CdtA from CdtB/CdtC must therefore precede the ATP-induced separation of CdtB from CdtC. CDT thus appears to use the physiology of the host endomembrane system as a sensing mechanism to regulate its two-stage disassembly. We predict ATP induces disassembly of the CdtB/CdtC dimer by acting as a competitive inhibitor of CdtB binding to CdtC. ATP does not affect the CDT holotoxin because CdtA either prevents ATP from binding to CdtB or stabilizes the interaction between CdtB and CdtC. In this project, we will establish the mechanism by which CdtA blocks the ATP-induced separation of CdtB from CdtC. We will identify the ATP biding site in CdtB by X-ray crystallography and NMR spectroscopy. This information will provide further insight into how ATP triggers CdtB/CdtC disassembly and will serve as a foundation for the generation of CdtB variants that cannot bind ATP. Those variants will then be used in a series of experiments to document how intracellular transport, disassembly, nuclear translocation, and cellular activity are affected when CDT contains a CdtB subunit that cannot bind ATP. Our work will elucidate both the how and why of the two-stage CDT disassembly process.
