title: The C-terminal region of Bacteroides fragilis toxin is essential to its biological activity.
abstract: To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.
authors.affiliation: Division of Infectious Diseases, Johns Hopkins University School of Medicine, Cancer Research Building II, 1550 Orleans Street, Loading Dock, Room 1M04, Baltimore, MD 21205, USA.
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Sears
authors.forename: Simy L
authors.initials: SL
authors.lastname: Buckwold
authors.forename: Jai W
authors.initials: JW
authors.lastname: Shin
authors.forename: Augusto A
authors.initials: AA
authors.lastname: Franco
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D015820:Cadherins / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D017384:Sequence Deletion
chemical_list: D001427:Bacterial Toxins; D015820:Cadherins; C405031:Bacteroides fragilis toxin; D008666:Metalloendopeptidases