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. 1999 Apr;44(4):504–510. doi: 10.1136/gut.44.4.504

Bacteroides fragilis toxin 2 damages human colonic mucosa in vitro

M Riegler 1, M Lotz 1, C Sears 1, C Pothoulakis 1, I Castagliuolo 1, C Wang 1, R Sedivy 1, T Sogukoglu 1, E Cosentini 1, G Bischof 1, W Feil 1, B Teleky 1, G Hamilton 1, J LaMont 1, E Wenzl 1
PMCID: PMC1727476  PMID: 10075957

Abstract

BACKGROUND—Strains of Bacteroides fragilis producing a 20 kDa protein toxin (B fragilis toxin (BFT) or fragilysin) are associated with diarrhoea in animals and humans. Although in vitro results indicate that BFT damages intestinal epithelial cells in culture, the effects of BFT on native human colon are not known. 
AIMS—To examine the electrophysiological and morphological effects of purified BFT-2 on human colonic mucosa in vitro. 
METHODS—For resistance (R) measurements, colonic mucosa mounted in Ussing chambers was exposed to luminal or serosal BFT-2 (1.25-10 nM) and after four hours morphological damage was measured on haematoxylin and eosin stained sections using morphometry. F actin distribution was assessed using confocal microscopy. 
RESULTS—Serosal BFT-2 for four hours was four-, two-, seven-, and threefold more potent than luminal BFT-2 in decreasing resistance, increasing epithelial 3H-mannitol permeability, and damaging crypt and surface colonocytes, respectively (p<0.05). Confocal microscopy showed reduced colonocyte F actin staining intensity after exposure to BFT-2. 
CONCLUSIONS—BFT-2 increases human colonic permeability and damages human colonic epithelial cells in vitro. These effects may be important in the development of diarrhoea and intestinal inflammation caused by B fragilis in vivo. 



Keywords: B fragilis toxin; toxin mediated colonocyte damage; actin filaments; transepithelial resistance; morphometry

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Figure 1 .

Figure 1

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Time course of the effect of BFT-2 on (A) colonic potential difference (PD), (B) short circuit current (Isc), and (C) resistance. Values represent mean (SEM); n = number of experiments. *p<0.05 versus controls; †p<0.05 versus luminal BFT. 


Figure 2 .

Figure 2

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Dose dependent effect of BFT-2 on colonic resistance. Values represent mean (SEM); n = number of experiments. *p<0.05 versus controls. 


Figure 3 .

Figure 3

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Effect of BFT-2 on colonic 3H-mannitol permeability. Values are mean (SEM) of five experiments performed in triplicate. *p<0.05, **p<0.01 versus controls. 


Figure 4 .

Figure 4

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Morphological effect of BFT-2 on human colonic mucosa. Human colonic mucosal sheets were incubated with either buffer alone for four hours (A) or serosal buffer containing 10 nM BFT-2 for 2.5 hours (B) or four hours (C, D). At the end of the incubation period sheets were fixed in formalin and processed for light microscopy. (A) Control tissue appears normal and epithelial cells form a continuous sheet over the lamina propria. (B) The mucosal sheet shows disruption of crypt epithelium whereas the surface epithelium remains intact. (C) The mucosal sheet shows disruption of surface and crypt epithelium. The arrowhead marks an area shown at higher magnification in (D) which shows rounding and detachment of crypt enterocytes into the lumen (long arrows). Surface colonocytes show cytoplasmic elongation and apical localisation of the pyknotic nucleus (arrowheads). Note loosening of the basal lamina (broad arrows). Original magnification × 140 (A, B); × 80 (C); × 420 (D). 


Figure 5 .

Figure 5

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Morphometric analysis of colonic epithelial cell damage caused by BFT-2. Values are mean (SEM). The number of experiments is given in brackets. *p<0.05 versus controls; †p<0.05 versus serosal 10 nM BFT-2. 


Figure 6 .

Figure 6

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Effect of BFT-2 on cellular F actin. Fluorescent photomicrograph of human colonic mucosal sheet incubated with buffer alone (A) or serosal buffer containing 10 nM BFT-2 for two hours (B) or four hours (C, D). Samples were fixed and fresh frozen sections cut perpendicularly (B) or vertically (A, C, D) to the long axis of surface cells. (A) Control cells had a polygonal shape with F actin distributed in the peripheral actin ring. Arrowheads mark intense F actin staining of the apical brush border. L, lumen; LP, lamina propria. (B) There was an overall reduction in F actin staining intensity and disruption of the F actin ring (arrowheads). (C) There was an overall reduction in F actin staining in epithelial cells (EC). The rectangle marks a crypt shown at higher magnification in (D), and the asterisk marks the basal lumen of the crypt. (D) Staining of the enterocyte F actin ring was nearly abolished. Some F actin staining remained in the brush border of damaged enterocytes (EC) or in exfoliated enterocytes (arrowheads). BL, basal lamina. Original magnification × 400 (A, B); × 200 (C); ×480 (D). 


Selected References

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

  1. Border M., Firehammer B. D., Shoop D. S., Myers L. L. Isolation of Bacteroides fragilis from the feces of diarrheic calves and lambs. J Clin Microbiol. 1985 Mar;21(3):472–473. doi: 10.1128/jcm.21.3.472-473.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chambers F. G., Koshy S. S., Saidi R. F., Clark D. P., Moore R. D., Sears C. L. Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro. Infect Immun. 1997 Sep;65(9):3561–3570. doi: 10.1128/iai.65.9.3561-3570.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Collins J. E., Bergeland M. E., Myers L. L., Shoop D. S. Exfoliating colitis associated with enterotoxigenic Bacteroides fragilis in a piglet. J Vet Diagn Invest. 1989 Oct;1(4):349–351. doi: 10.1177/104063878900100413. [DOI] [PubMed] [Google Scholar]
  4. Dillon S. T., Rubin E. J., Yakubovich M., Pothoulakis C., LaMont J. T., Feig L. A., Gilbert R. J. Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B. Infect Immun. 1995 Apr;63(4):1421–1426. doi: 10.1128/iai.63.4.1421-1426.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Duimstra J. R., Myers L. L., Collins J. E., Benfield D. A., Shoop D. S., Bradbury W. C. Enterovirulence of enterotoxigenic Bacteroides fragilis in gnotobiotic pigs. Vet Pathol. 1991 Nov;28(6):514–518. doi: 10.1177/030098589102800608. [DOI] [PubMed] [Google Scholar]
  7. Fasano A., Fiorentini C., Donelli G., Uzzau S., Kaper J. B., Margaretten K., Ding X., Guandalini S., Comstock L., Goldblum S. E. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest. 1995 Aug;96(2):710–720. doi: 10.1172/JCI118114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feil W., Lacy E. R., Wong Y. M., Burger D., Wenzl E., Starlinger M., Schiessel R. Rapid epithelial restitution of human and rabbit colonic mucosa. Gastroenterology. 1989 Sep;97(3):685–701. doi: 10.1016/0016-5085(89)90640-9. [DOI] [PubMed] [Google Scholar]
  9. Franco A. A., Mundy L. M., Trucksis M., Wu S., Kaper J. B., Sears C. L. Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene. Infect Immun. 1997 Mar;65(3):1007–1013. doi: 10.1128/iai.65.3.1007-1013.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grass G. M., Sweetana S. A. In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharm Res. 1988 Jun;5(6):372–376. doi: 10.1023/a:1015911712079. [DOI] [PubMed] [Google Scholar]
  11. Just I., Fritz G., Aktories K., Giry M., Popoff M. R., Boquet P., Hegenbarth S., von Eichel-Streiber C. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J Biol Chem. 1994 Apr 8;269(14):10706–10712. [PubMed] [Google Scholar]
  12. Just I., Selzer J., Wilm M., von Eichel-Streiber C., Mann M., Aktories K. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature. 1995 Jun 8;375(6531):500–503. doi: 10.1038/375500a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Myers L. L., Shoop D. S. Association of enterotoxigenic Bacteroides fragilis with diarrheal disease in young pigs. Am J Vet Res. 1987 May;48(5):774–775. [PubMed] [Google Scholar]
  16. Myers L. L., Shoop D. S., Byars T. D. Diarrhea associated with enterotoxigenic Bacteroides fragilis in foals. Am J Vet Res. 1987 Nov;48(11):1565–1567. [PubMed] [Google Scholar]
  17. 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]
  18. Myers L. L., Shoop D. S., Collins J. E. Rabbit model to evaluate enterovirulence of Bacteroides fragilis. J Clin Microbiol. 1990 Jul;28(7):1658–1660. doi: 10.1128/jcm.28.7.1658-1660.1990. [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, Azghani A. O., Wilkins T. D. The Bacteroides fragilis toxin fragilysin disrupts the paracellular barrier of epithelial cells. Infect Immun. 1997 Apr;65(4):1431–1439. doi: 10.1128/iai.65.4.1431-1439.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Pantosti A., Piersimoni C., Perissi G. Detection of Bacteroides fragilis enterotoxin in the feces of a child with diarrhea. Clin Infect Dis. 1994 Oct;19(4):809–810. doi: 10.1093/clinids/19.4.809. [DOI] [PubMed] [Google Scholar]
  24. Riegler M., Sedivy R., Pothoulakis C., Hamilton G., Zacherl J., Bischof G., Cosentini E., Feil W., Schiessel R., LaMont J. T. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. J Clin Invest. 1995 May;95(5):2004–2011. doi: 10.1172/JCI117885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Riegler M., Sedivy R., Sogukoglu T., Cosentini E., Bischof G., Teleky B., Feil W., Schiessel R., Hamilton G., Wenzl E. Epidermal growth factor promotes rapid response to epithelial injury in rabbit duodenum in vitro. Gastroenterology. 1996 Jul;111(1):28–36. doi: 10.1053/gast.1996.v111.pm8698221. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Saidi R. F., Jaeger K., Montrose M. H., Wu S., Sears C. L. Bacteroides fragilis toxin rearranges the actin cytoskeleton of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content. Cell Motil Cytoskeleton. 1997;37(2):159–165. doi: 10.1002/(SICI)1097-0169(1997)37:2<159::AID-CM8>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. Wells C. L., van de Westerlo E. M., Jechorek R. P., Feltis B. A., Wilkins T. D., Erlandsen S. L. Bacteroides fragilis enterotoxin modulates epithelial permeability and bacterial internalization by HT-29 enterocytes. Gastroenterology. 1996 May;110(5):1429–1437. doi: 10.1053/gast.1996.v110.pm8613048. [DOI] [PubMed] [Google Scholar]

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