B-155. Identifying the Molecular Basis of Cytolethal Distending Toxin Binding to Mammalian Cells

A. Gargi1, M. G. Prouty1, A. Eshraghi2, F. J. Maldonado2, K. A. Bradley2, S. R. Blanke1;
1Univ. of Illinois, Urbana, IL, 2Univ. of California, Los Angeles, CA.

The cytolethal distending toxins (CDTs) comprise an important family of genotoxic cyclomodulins that are produced by an expanding number of Gram-negative pathogens that occupy disparate niches within the host. However, the molecular basis of CDT interactions with host cells is poorly defined. Analogous to other intracellular-acting bacterial toxins, CDTs possess an “AB” architecture, where the B-moiety facilitates the entry of the catalytic A-moiety into sensitive cells. However, CDTs are comprised of three non-covalently interacting proteins - CdtA, CdtB, and CdtC, indicating structure-function relationships that are idiosyncratic to this family of toxins. The objective of this study was to evaluate the molecular basis by which CDTs interact with mammalian cells. Based on high-resolution structural data for the Haemophilus ducreyi CDT, the accessible surfaces of CdtA and CdtC were probed for their roles in cell binding by scanning different topological domains using site-directed mutagenesis. Wild type and mutant forms of the H. ducreyi CdtA and CdtC were prepared, and expressed as recombinant proteins in E. coli. Each of these proteins were evaluated for proper folding, assembly into holotoxin, the capacity to induce G2/M cell cycle arrest, and to bind the surface of cultured mammalian cells. The studies identified three residues in a newly identified region in CdtA that were critical for binding. Conversion of these residues to alanine attenuated cell cycle arrest activity by greater than 1000-fold, and resulted in undetectable binding to the cell surface. These results provide new information about specific residues most important for CDT cell recognition and binding, as well as new insights into the role of CdtA for the function of the holotoxin. Understanding the molecular basis of CDT cellular affinity and specificity will be critical for not only understanding the mechanism of cellular intoxication, but will also reveal strategies for antagonizing interactions of these toxins with host cells.