title: Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.
abstract: Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Franco
authors.forename: L M
authors.initials: LM
authors.lastname: Mundy
authors.forename: M
authors.initials: M
authors.lastname: Trucksis
authors.forename: S
authors.initials: S
authors.lastname: Wu
authors.forename: J B
authors.initials: JB
authors.lastname: Kaper
authors.forename: C L
authors.initials: CL
authors.lastname: Sears
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics*; D001441:Bacteroides fragilis / Q000235:genetics / Q000472:pathogenicity*; D001483:Base Sequence; D003001:Cloning, Molecular; D018628:Gene Dosage; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013552:Swine
chemical_list: D001427:Bacterial Toxins; D008666:Metalloendopeptidases