Induction of persistent colitis by a human commensal, enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice

Abstract

Enterotoxigenic Bacteroides fragilis (ETBF) causes diarrhea and is implicated in inflammatory bowel diseases and colorectal cancer. The only known ETBF virulence factor is the Bacteroides fragilis toxin (BFT), which induces E-cadherin cleavage, interleukin-8 secretion, and epithelial cell proliferation. A murine model for ETBF has not been characterized. Specific pathogen-free (SPF) C57BL/6J or germfree 129S6/SvEv mice were orally inoculated with wild-type ETBF (WT-ETBF) strains, a nontoxigenic WT strain of B. fragilis (WT-NTBF), WT-NTBF overexpressing bft (rETBF), or WT-NTBF overexpressing a biologically inactive mutated bft (rNTBF). In SPF and germfree mice, ETBF caused colitis but was lethal only in germfree mice. Colonic histopathology demonstrated mucosal thickening with inflammatory cell infiltration, crypt abscesses, and epithelial cell exfoliation, erosion, and ulceration. SPF mice colonized with rETBF mimicked WT-ETBF, whereas rNTBF caused no histopathology. Intestinal epithelial E-cadherin was rapidly cleaved in vivo in WT-ETBF-colonized mice and in vitro in intestinal tissues cultured with purified BFT. ETBF mice colonized for 16 months exhibited persistent colitis. BFT did not directly induce lymphocyte proliferation, dendritic cell stimulation, or Toll-like receptor activation. In conclusion, WT-ETBF induced acute then persistent colitis in SPF mice and rapidly lethal colitis in WT germfree mice. Our data support the hypothesis that chronic colonization with the human commensal ETBF can induce persistent, subclinical colitis in humans.

FIG. 1.

Clinicopathologic analysis of C57BL/6 mice inoculated with B. fragilis (∼1 × 10⁹ CFU) for 7 days. (a) Weight change. The daily body weight of individual mice was normalized to the starting body weight. Shown are pooled data from three independent experiments, with ∼4 to 15 mice per experiment. The baseline weight (in grams) of individual mice was similar between the three mouse groups as follows: mock-infected mice (Mock), 14.42 ± 0.35 (standard error of the mean); WT-NTBF-infected mice, 14.62 ± 0.43; and WT-ETBF (bft-2)-infected mice, 14.47 ± 0.42. *, a P value of ≤0.001 for WT-ETBF (bft-2)-infected mice compared with either WT-NTBF- or mock-infected mice, except for day 1 which is significant (P < 0.005) only compared with mock-infected mice; #, a P value of <0.01 for WT-NTBF-infected mice compared with mock-infected mice. (b) Mortality of WT-ETBF (bft-2)-infected (n = 55), WT-NTBF-infected (n = 44), or mock-infected (n = 35) mice. Shown are pooled data from seven independent experiments. (c) Cecal weight and gross morphology (a total of ∼5 to 7 mice) (inset). (d) Spleen weight and gross morphology (a total of ∼5 to 7 mice) (inset). Bars, median weight. NS, statistically not significant.

FIG. 2.

ETBF induces acute colitis. H&E-stained tissue sections of mock-, WT-NTBF-, WT-ETBF (bft-2)-, and rETBF (bft-2)-infected mice for 2 days (a) and 7 days (b). Boxed areas were digitally magnified (×100). Bars, 100 μm. (c) Histologic inflammation scores of large bowels of infected mice at day 1, day 2, day 3, and day 7. Results of mock-infected mice were identical to those of WT-NTBF-infected mice (data not shown). Asterisks denote a lack of inflammation (an inflammation score of <0.2). (d) H&E-stained cecal tissue sections from BT4001- and BT4001 (bft-2)-infected mice.

FIG. 3.

Western blot analysis of mouse serum against B. fragilis (B.f.) lysates. Shown are pooled sera (1:500 dilution) from mock-infected (Mock), WT-NTBF-infected, or WT-ETBF (bft-2)-infected mice evaluated on B. fragilis lysates. Films were exposed for 10 s and 1 h.

FIG. 4.

ETBF induces E-cadherin cleavage in vitro and ex vivo. (a) Uninfected mouse small intestine (Si), cecum (Ce), and mid-colon (Co) tissues cultured with (+) or without (−) purified BFT (5 nM) for 15 min and probed for E-cadherin (N terminus specific) in the lysate and the supernatant. Results are representative of three independent experiments. Pan-cytokeratin was used as a CEC loading control, and GAPDH was used as a total protein loading control. (b) Mice were infected for 12, 18, and 24 h, and the ceca were cut lengthwise into two pieces. (c) One piece was used for Western blot analysis and the second half was used for H&E sections. Results are representative of two independent experiments (a total of three mice per experiment).

FIG. 5.

ETBF chronic infection enhances epithelial hyperplasia. (a) H&E staining and anti-cyclin D1 immunohistochemistry of ceca from mice persistently infected (for 1 month) with WT-NTBF and WT-ETBF (bft-2). Results for mock-infected mice are identical to those for WT-NTBF-infected mice (data not shown). (b) H&E staining of ceca from mice persistently infected for 8 months [WT-NTBF and WT-ETBF (bft-2)] and 16 months [WT-NTBF and rETBF(bft-2)].

FIG. 6.

ETBF induces colitis in germfree mice. Germfree 129S6/SvEv mice (∼4 to 10 months old) were inoculated with WT-ETBF (bft-2), rETBF (bft-2), or rNTBF (bft-2 H352Y), and ceca were prepared for histology 3 days later. Bars, 100 μm.