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Infection and Immunity, May 2002, p. 2463-2471, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2463-2471.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Diversity of the Metalloprotease Toxin Produced by Enterotoxigenic Bacteroides fragilis

Shaoguang Wu,1,2 Lawrence A. Dreyfus,3 Art O. Tzianabos,4 Chika Hayashi,1,2 and Cynthia L. Sears1,2*

Divisions of Infectious Diseases,1 Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,2 Division of Cell Biology and Biophysics, University of Missouri—Kansas City, Kansas City, Missouri 64110,3 Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 021154

Received 24 July 2001/ Returned for modification 20 September 2001/ Accepted 14 February 2002

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a 20-kDa zinc metalloprotease toxin (BFT) associated with diarrheal disease of animals, young children, and adults. BFT stimulates secretion in intestinal loops in vivo and modifies epithelial cell morphology in vitro. The B. fragilis toxin (bft) gene from ETBF strain 86-5443-2-2 (piglet; bft-2) revealed significant nucleotide and predicted amino acid differences when compared to the bft gene from ETBF strain VPI 13784 (lamb; bft-1). This study compares BFT-1 and BFT-2, respectively, produced by ETBF strains VPI 13784 and 86-5443-2-2 purified using the Van Tassell method (38) and a modified purification scheme described herein. Multiple differences in the protein toxins produced by these ETBF strains were identified. First, purified BFT-1 eluted from a high-resolution anion-exchange column (Mono Q) at 0.22 ± 0.005 M NaC1 versus 0.18 ± 0.001 M NaC1 for BFT-2 (P < 0.001). Second, BFT-1 and BFT-2 exhibited different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase fast protein liquid chromatography. Third, each BFT reacted with greater specificity to homologous rather than heterologous antisera. Fourth, BFT-2 had modest, but consistently, greater biological activity than BFT-1 when tested on HT29/C1 cells (P <= 0.01). Together, these data indicate that these ETBF strains produce two distinct isotypes of BFT, termed BFT-1 (VPI 13784 BFT) and BFT-2 (86-5443-2-2 BFT) to recognize the order in which the proteins were purified and genetic sequences identified. The modified purification scheme described in this report yields about two to three times more purified BFT protein than previous protocols and is less time consuming.

* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross Building, Room 1167, Baltimore, MD 21205. Phone: (410) 614-0141. Fax: (410) 955-7889. E-mail: csears{at}jhmi.edu.

Editor: J. T. Barbieri

Infection and Immunity, May 2002, p. 2463-2471, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2463-2471.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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