title: 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 association 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 proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] 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.
authors.affiliation: Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Boesze-Battaglia
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Lisa P
authors.initials: LP
authors.lastname: Walker
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Zekavat
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Mensur
authors.initials: M
authors.lastname: Dlakić
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Monika Damek
authors.initials: MD
authors.lastname: Scuron
authors.affiliation: Divisions of Molecular Surface Physics and Nanoscience and Molecular Physics, Linköping University, Linköping, Sweden.
authors.forename: Patrik
authors.initials: P
authors.lastname: Nygren
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA shenker@upenn.edu.
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Shenker
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D016871:Pasteurellaceae Infections / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; C067044:cytolethal distending toxin; D002784:Cholesterol