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First published online 15 May 2007
doi: 10.1242/jcs.03455

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Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and -secretase-dependent E-cadherin cleavage

¹ Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, 1550 Orleans St, CRB2 Bldg Suite 1M.05, Baltimore, MD 21231, USA
² Division of Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, 1550 Orleans St, CRB2 Bldg Suite 1M.05, Baltimore, MD 21231, USA

View larger version (36K): [in this window] [in a new window]   Fig. 1. BFT induces release of the E-cadherin ectodomain and shedding of IEC membrane proteins. (A) BFT induces release of the E-cadherin ectodomain. Upper panel. Cell lysates of HT29/C1 cells treated with BFT (5 nM) for 0.5 hour to 3 hours were evaluated by western blot using the E2 antibody that recognizes the C-terminal domain of E-cadherin. Actin served as an internal control for protein loading. Blot is representative of five experiments. Lower panel. TCA-precipitated cell culture supernatants of HT29/C1 cells treated with BFT (5 nM) for 0.5 hour to 3 hours were evaluated by western blot using the H108 antibody that recognizes the ectodomain of E-cadherin. Transferrin, detected by Coomasie Blue staining, served as an internal control for protein loading. Blot is representative of three experiments. (B) Normalized western blot data demonstrating significant BFT-induced cleavage of intact E-cadherin (120 kDa) on HT29/C1 cells by 0.5 hour (P<0.01) with enhanced detection of a 33 kDa cell-associated E-cadherin fragment at 0.5 hour and release of the 80 kDa E-cadherin ectodomain into cell supernatants (both P<0.001). The 33 kDa E-cadherin fragment is degraded over time (see also Fig. 1A). Data are means ± s.d. of three experiments. (C) BFT (5 nM, 1 hour) induces shedding of IEC membrane proteins as well as the E-cadherin ectodomain (80 kDa). HT29/C1 cell membrane proteins were biotin-labeled and processed as in the Materials and Methods. Transferrin stained by Coomasie Blue serves as an internal control for protein loading. Blots are representative of four experiments. (D) Cleavage of E-cadherin extracellular domain requires biologically active BFT. HT29/C1 cells were treated with purified BFT (5 nM) or culture supernatants of B. fragilis 9343(pFD340::P-bft) that expresses wild-type BFT or B. fragilis 9343(pFD340::P-bftH352Y) that expresses mutant biologically inactive BFT. Cell lysates and TCA-precipitated cell culture supernatants were assessed by western blot using the E2 (upper lane) and H108 (lower lane) antibodies to the E-cadherin C-terminus or ectodomain, respectively. Lane 1, untreated control; lane 2 purified BFT; lane 3, B. fragilis 9343(pFD340::P-bft); lane 4, B. fragilis 9343(pFD340::P-bftH352Y); lane 5, brain heart infusion broth alone.

View larger version (38K): [in this window] [in a new window]   Fig. 2. MMP-7, ADAM10 or ADAM17 do not mediate BFT-initiated E-cadherin ectodomain shedding whereas -secretase mediates, in part, BFT-initiated E-cadherin cleavage. Cell lysates of control or siRNA-treated [MMP-7 (A), ADAM10 (B), ADAM17 (C) or -secretase (D) ribonucleotide pairs] HT29/C1 cells were examined by western blot for each target protein or E-cadherin (intact 120 kDa or 33 kDa degradation fragment) in the presence or absence of BFT (5 nM, 2 hours) treatment. -actin served as an internal protein loading control. Blots are representative of three experiments.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Inhibition of -secretase, but not -secretase, blocks BFT-induced cleavage of the C-terminal domain of E-cadherin and partially -catenin redistribution. (A) Cell lysates of HT29/C1 cells were analyzed by western blot using the E2 antibody to the C-terminus of E-cadherin. Cells were treated with BFT alone (30 minutes, 5 nM) or with inhibitors [-secretase inhibitor, AEBSF (0.5 mM) or -secretase inhibitor, L-685,458 (1.5 µM)] for 30 minutes prior to BFT treatment. Molecular size markers indicate the 120 kDa intact E-cadherin and the 33 and 28 kDa degradation fragments of E-cadherin. -catenin serves as an internal control for protein loading. Blot is representative of three experiments. (B) HT29/C1 cells were treated with BFT alone (5 nM) or in the presence of the -secretase inhibitor, L-685,458 (1.5 µM), followed by immunostaining with an anti--catenin antibody (CAT-5H10) (green) and the E2 antibody to the C-terminus of E-cadherin (red). Untreated control cells or cells pretreated for 30 minutes with L-685,458 (Panels a and e, respectively) reveal co-association (yellow) of E-cadherin and -catenin staining. After BFT treatment, there is diffusion and dissociation of E-cadherin and -catenin staining at 1 hour (Panel b) with progressive cytoplasmic -catenin and E-cadherin diffusion over the subsequent 3- and 6-hour time points (Panels c,d). In cells pretreated with L-685,458, there is also dissociation of E-cadherin and -catenin staining at 1 hour but the -catenin signal is intense and localized at the cell membrane (Panel f). Over time, partial cytoplasmic diffusion of -catenin occurs but with retention of a distinct -catenin pool on the cell membrane (panels g,h). Representative of three experiments. (C) -catenin/actin ratios in control HT29/C1 cells or cells treated with BFT (5 nM) for 6 hours in the presence or absence of L-685,458 (1.5 µM). L+B6h=L-685,458 + BFT for 6 hours. P<0.05, Control vs BFT, 6 hours. Data are means ± s.d. of five experiments (D) HT29/C1 cells were treated with BFT (5 nM) for 1, 3 or 6 hours in the presence or absence of L-685,458 (1.5 µM). Cell lysates were immunoprecipitated using an antibody to -secretase and the western blot analyzed using antibodies to the C-terminus of E-cadherin (E2) (upper panel), -catenin (CAT-5H10) (middle panel) or -secretase (lower panel). Intact E-cadherin is 120 kDa. -secretase inhibition results in stable association of a 33 kDa C-terminal E-cadherin fragment and -catenin with -secretase. Western blot is representative of two experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Effect of -secretase inhibition on basal and BFT-induced cell proliferation. Cell proliferation in parental HT29/C1 cells and TCF dominant negative HT29/C1 cells (HT29/C1::TcfN31). Cell lines were treated with BFT (5 nM) alone or in the presence of the -secretase inhibitor, L685,458 (1.5 µM). Results are depicted as changes in the percentage uptake of [3H]thymidine compared with control cells for each cell line. BFT induces a significant increase in HT29/C1 cell proliferation in both parental and TCF dominant negative HT29/C1 cell lines (lanes 1 or 4, P<0.001 for both cell lines vs. control) that is unaltered by L685,458 (comparisons lanes 1 vs 3 and lanes 4 vs 6, P=NS). By contrast, L685,458 inhibits parental HT29/C1 cell proliferation (lane 2, P<0.001) that is ablated in TCF dominant negative HT29/C1 cells (lane 5, P<0.01 compared with lane 2) indicating -secretase regulation of parental HT29/C1 cell proliferation is dependent on -catenin/TCF nuclear signaling. Data are means ± s.d. of 3-4 experiments conducted with 3-5 replicates per experiment.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Proposed model of BFT mechanism of action. BFT binds to and probably cleaves a specific host cell receptor (Wu et al., 2006), potentially activating a host cell protease that either alone or complexed with BFT then cleaves the E-cadherin ectodomain and stimulates activation of -secretase that cleaves the cell-associated E-cadherin remnant. Alternatively, BFT receptor binding may stimulate signal transduction activating a host cell protease that stimulates cleavage of E-cadherin.

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