title: Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.
abstract: Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S1A8.
authors.forename: Zhifen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Minyoung
authors.initials: M
authors.lastname: Park
authors.forename: John
authors.initials: J
authors.lastname: Tam
authors.forename: Anick
authors.initials: A
authors.lastname: Auger
authors.forename: Greg L
authors.initials: GL
authors.lastname: Beilhartz
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
authors.forename: Roman A
authors.initials: RA
authors.lastname: Melnyk
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D016360:Clostridioides difficile / Q000235:genetics / Q000472:pathogenicity*; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012414:Rubidium Radioisotopes / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; C488583:Clostridium difficile lethal toxin B; D052899:Pore Forming Cytotoxic Proteins; D012414:Rubidium Radioisotopes