Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Mar;65(3):1007–1013. doi: 10.1128/iai.65.3.1007-1013.1997

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

A A Franco 1, L M Mundy 1, M Trucksis 1, S Wu 1, J B Kaper 1, C L Sears 1
PMCID: PMC175082  PMID: 9038310

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).

Full Text

The Full Text of this article is available as a PDF (195.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Donelli G., Fabbri A., Fiorentini C. Bacteroides fragilis enterotoxin induces cytoskeletal changes and surface blebbing in HT-29 cells. Infect Immun. 1996 Jan;64(1):113–119. doi: 10.1128/iai.64.1.113-119.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gorbach S. L., Bartlett J. G. Anaerobic infections. 1. N Engl J Med. 1974 May 23;290(21):1177–1184. doi: 10.1056/NEJM197405232902106. [DOI] [PubMed] [Google Scholar]
  4. Huttner W. B. Cell biology. Snappy exocytoxins. Nature. 1993 Sep 9;365(6442):104–105. doi: 10.1038/365104a0. [DOI] [PubMed] [Google Scholar]
  5. Häse C. C., Finkelstein R. A. Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev. 1993 Dec;57(4):823–837. doi: 10.1128/mr.57.4.823-837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kapral F. A. Staphylococcus aureus delta toxin as an enterotoxin. Ciba Found Symp. 1985;112:215–229. doi: 10.1002/9780470720936.ch12. [DOI] [PubMed] [Google Scholar]
  7. Kato N., Karuniawati A., Jotwani R., Kato H., Watanabe K., Ueno K. Isolation of enterotoxigenic Bacteroides fragilis from extraintestinal sites by cell culture assay. Clin Infect Dis. 1995 Jun;20 (Suppl 2):S141–S141. doi: 10.1093/clinids/20.supplement_2.s141. [DOI] [PubMed] [Google Scholar]
  8. Kerr I. D., Dufourcq J., Rice J. A., Fredkin D. R., Sansom M. S. Ion channel formation by synthetic analogues of staphylococcal delta-toxin. Biochim Biophys Acta. 1995 Jun 14;1236(2):219–227. doi: 10.1016/0005-2736(95)00051-4. [DOI] [PubMed] [Google Scholar]
  9. Koshy S. S., Montrose M. H., Sears C. L. Human intestinal epithelial cells swell and demonstrate actin rearrangement in response to the metalloprotease toxin of Bacteroides fragilis. Infect Immun. 1996 Dec;64(12):5022–5028. doi: 10.1128/iai.64.12.5022-5028.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lencer W. I., de Almeida J. B., Moe S., Stow J. L., Ausiello D. A., Madara J. L. Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation. J Clin Invest. 1993 Dec;92(6):2941–2951. doi: 10.1172/JCI116917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McCombie W. R., Heiner C., Kelley J. M., Fitzgerald M. G., Gocayne J. D. Rapid and reliable fluorescent cycle sequencing of double-stranded templates. DNA Seq. 1992;2(5):289–296. doi: 10.3109/10425179209030961. [DOI] [PubMed] [Google Scholar]
  12. McIver K. S., Kessler E., Olson J. C., Ohman D. E. The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol. 1995 Dec;18(5):877–889. doi: 10.1111/j.1365-2958.1995.18050877.x. [DOI] [PubMed] [Google Scholar]
  13. Mellor I. R., Thomas D. H., Sansom M. S. Properties of ion channels formed by Staphylococcus aureus delta-toxin. Biochim Biophys Acta. 1988 Jul 21;942(2):280–294. doi: 10.1016/0005-2736(88)90030-2. [DOI] [PubMed] [Google Scholar]
  14. Miller J. R., Barrett L. J., Kotloff K., Guerrant R. L. A rapid test for infectious and inflammatory enteritis. Arch Intern Med. 1994 Dec 12;154(23):2660–2664. doi: 10.1001/archinte.1994.00420230043006. [DOI] [PubMed] [Google Scholar]
  15. Moncrief J. S., Obiso R., Jr, Barroso L. A., Kling J. J., Wright R. L., Van Tassell R. L., Lyerly D. M., Wilkins T. D. The enterotoxin of Bacteroides fragilis is a metalloprotease. Infect Immun. 1995 Jan;63(1):175–181. doi: 10.1128/iai.63.1.175-181.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mundy L. M., Sears C. L. Detection of toxin production by Bacteroides fragilis: assay development and screening of extraintestinal clinical isolates. Clin Infect Dis. 1996 Aug;23(2):269–276. doi: 10.1093/clinids/23.2.269. [DOI] [PubMed] [Google Scholar]
  17. Myers L. L., Firehammer B. D., Shoop D. S., Border M. M. Bacteroides fragilis: a possible cause of acute diarrheal disease in newborn lambs. Infect Immun. 1984 May;44(2):241–244. doi: 10.1128/iai.44.2.241-244.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Myers L. L., Shoop D. S., Collins J. E., Bradbury W. C. Diarrheal disease caused by enterotoxigenic Bacteroides fragilis in infant rabbits. J Clin Microbiol. 1989 Sep;27(9):2025–2030. doi: 10.1128/jcm.27.9.2025-2030.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Myers L. L., Shoop D. S., Firehammer B. D., Border M. M. Association of enterotoxigenic Bacteroides fragilis with diarrheal disease in calves. J Infect Dis. 1985 Dec;152(6):1344–1347. doi: 10.1093/infdis/152.6.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myers L. L., Shoop D. S., Stackhouse L. L., Newman F. S., Flaherty R. J., Letson G. W., Sack R. B. Isolation of enterotoxigenic Bacteroides fragilis from humans with diarrhea. J Clin Microbiol. 1987 Dec;25(12):2330–2333. doi: 10.1128/jcm.25.12.2330-2333.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Obiso R. J., Jr, Lyerly D. M., Van Tassell R. L., Wilkins T. D. Proteolytic activity of the Bacteroides fragilis enterotoxin causes fluid secretion and intestinal damage in vivo. Infect Immun. 1995 Oct;63(10):3820–3826. doi: 10.1128/iai.63.10.3820-3826.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Polk B. F., Kasper D. L. Bacteroides fragilis subspecies in clinical isolates. Ann Intern Med. 1977 May;86(5):569–571. doi: 10.7326/0003-4819-86-5-569. [DOI] [PubMed] [Google Scholar]
  23. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sack R. B., Albert M. J., Alam K., Neogi P. K., Akbar M. S. Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi children with diarrhea: a controlled study. J Clin Microbiol. 1994 Apr;32(4):960–963. doi: 10.1128/jcm.32.4.960-963.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sack R. B., Myers L. L., Almeido-Hill J., Shoop D. S., Bradbury W. C., Reid R., Santosham M. Enterotoxigenic Bacteroides fragilis: epidemiologic studies of its role as a human diarrhoeal pathogen. J Diarrhoeal Dis Res. 1992 Mar;10(1):4–9. [PubMed] [Google Scholar]
  26. Saidi R. F., Sears C. L. Bacteroides fragilis toxin rapidly intoxicates human intestinal epithelial cells (HT29/C1) in vitro. Infect Immun. 1996 Dec;64(12):5029–5034. doi: 10.1128/iai.64.12.5029-5034.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. San Joaquin V. H., Griffis J. C., Lee C., Sears C. L. Association of Bacteroides fragilis with childhood diarrhea. Scand J Infect Dis. 1995;27(3):211–215. doi: 10.3109/00365549509019011. [DOI] [PubMed] [Google Scholar]
  28. Schiavo G., Benfenati F., Poulain B., Rossetto O., Polverino de Laureto P., DasGupta B. R., Montecucco C. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992 Oct 29;359(6398):832–835. doi: 10.1038/359832a0. [DOI] [PubMed] [Google Scholar]
  29. Sears C. L., Myers L. L., Lazenby A., Van Tassell R. L. Enterotoxigenic Bacteroides fragilis. Clin Infect Dis. 1995 Jun;20 (Suppl 2):S142–S148. doi: 10.1093/clinids/20.supplement_2.s142. [DOI] [PubMed] [Google Scholar]
  30. Sukumar M., Rizo J., Wall M., Dreyfus L. A., Kupersztoch Y. M., Gierasch L. M. The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism. Protein Sci. 1995 Sep;4(9):1718–1729. doi: 10.1002/pro.5560040907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trucksis M., Galen J. E., Michalski J., Fasano A., Kaper J. B. Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5267–5271. doi: 10.1073/pnas.90.11.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Van Tassel R. L., Lyerly D. M., Wilkins T. D. Production of antisera against the enterotoxin of Bacteroides fragilis and their use in a cytotoxicity neutralization assay of HT-29 cells. Clin Diagn Lab Immunol. 1994 Jul;1(4):473–476. doi: 10.1128/cdli.1.4.473-476.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Van Tassell R. L., Lyerly D. M., Wilkins T. D. Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 1994 Sep;1(5):578–584. doi: 10.1128/cdli.1.5.578-584.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Van Tassell R. L., Lyerly D. M., Wilkins T. D. Purification and characterization of an enterotoxin from Bacteroides fragilis. Infect Immun. 1992 Apr;60(4):1343–1350. doi: 10.1128/iai.60.4.1343-1350.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Viscidi R., Willey S., Bartlett J. G. Isolation rates and toxigenic potential of Clostridium difficile isolates from various patient populations. Gastroenterology. 1981 Jul;81(1):5–9. [PubMed] [Google Scholar]
  36. Weikel C. S., Grieco F. D., Reuben J., Myers L. L., Sack R. B. Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology. Infect Immun. 1992 Feb;60(2):321–327. doi: 10.1128/iai.60.2.321-327.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wright A. C., Guo Y., Johnson J. A., Nataro J. P., Morris J. G., Jr Development and testing of a nonradioactive DNA oligonucleotide probe that is specific for Vibrio cholerae cholera toxin. J Clin Microbiol. 1992 Sep;30(9):2302–2306. doi: 10.1128/jcm.30.9.2302-2306.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES