The Aggregatibacter actinomycetemcomitans cytolethal distending toxin active subunit, CdtB, contains a cholesterol recognition sequence required for toxin binding and subunit internalization

Abstract

Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the associaton of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the pro-inflammatory response in macrophages (IL-1β and TNFα release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.