Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene

Augusto A. Franco, Linda M. Mundy, Michele Trucksis, Shaoguang Wu, James B. Kaper, Cynthia Louise Sears

Research output: Contribution to journalArticle

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).

Original languageEnglish (US)
Pages (from-to)1007-1013
Number of pages7
JournalInfection and Immunity
Volume65
Issue number3
StatePublished - 1997

Fingerprint

Metalloproteases
Organism Cloning
Genes
Bacteroides fragilis
Bacteroides fragilis toxin
Animal Diseases
Oligonucleotide Probes
Gelatin

ASJC Scopus subject areas

  • Immunology

Cite this

Franco, A. A., Mundy, L. M., Trucksis, M., Wu, S., Kaper, J. B., & Sears, C. L. (1997). Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene. Infection and Immunity, 65(3), 1007-1013.

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene. / Franco, Augusto A.; Mundy, Linda M.; Trucksis, Michele; Wu, Shaoguang; Kaper, James B.; Sears, Cynthia Louise.

In: Infection and Immunity, Vol. 65, No. 3, 1997, p. 1007-1013.

Research output: Contribution to journalArticle

Franco, AA, Mundy, LM, Trucksis, M, Wu, S, Kaper, JB & Sears, CL 1997, 'Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene', Infection and Immunity, vol. 65, no. 3, pp. 1007-1013.
Franco, Augusto A. ; Mundy, Linda M. ; Trucksis, Michele ; Wu, Shaoguang ; Kaper, James B. ; Sears, Cynthia Louise. / Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene. In: Infection and Immunity. 1997 ; Vol. 65, No. 3. pp. 1007-1013.
@article{8eda72d3bfb845e0a4c116b762b62632,
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).",
author = "Franco, {Augusto A.} and Mundy, {Linda M.} and Michele Trucksis and Shaoguang Wu and Kaper, {James B.} and Sears, {Cynthia Louise}",
year = "1997",
language = "English (US)",
volume = "65",
pages = "1007--1013",
journal = "Infection and Immunity",
issn = "0019-9567",
publisher = "American Society for Microbiology",
number = "3",

}

TY - JOUR

T1 - Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene

AU - Franco, Augusto A.

AU - Mundy, Linda M.

AU - Trucksis, Michele

AU - Wu, Shaoguang

AU - Kaper, James B.

AU - Sears, Cynthia Louise

PY - 1997

Y1 - 1997

N2 - 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).

AB - 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).

UR - http://www.scopus.com/inward/record.url?scp=0031027277&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031027277&partnerID=8YFLogxK

M3 - Article

C2 - 9038310

AN - SCOPUS:0031027277

VL - 65

SP - 1007

EP - 1013

JO - Infection and Immunity

JF - Infection and Immunity

SN - 0019-9567

IS - 3

ER -