Bacterial strains and culture conditions
The strains used in this study were L. casei ATCC 27092 and E. coli DH5α. L. casei was grown at 37°C in MRS broth to produce the recombinant strain or at 30°C in MRS/K (MRS with 0.2 M potassium phosphate buffer) to produce the cell culture supernatant . Erythromycin (5 μg/ml) was added to MRS or MRS/K to select the recombinant strain. E. coli was grown at 37°C in LB medium with or without ampicillin (100 μg/ml).
Construction of the rCTB–YVAD secretion vector
PCR was performed using the plasmid pSCTB  as the template DNA, KOD-Plus- DNA polymerase (Toyobo, Osaka, Japan), and primers containing the YVAD-coding sequence fused to the C-terminus of the CTB gene: sense, 5′-caccaccaccaccaccactaaaggccttc-3′; anti-sense, 5′-atcagcaacataatttgccatactaattgc-3′. Initial denaturation was at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec, and extension at 68°C for 7 min. The DNA fragment of about 6,600 bp was separated by agarose gel electrophoresis and extracted from the gel with the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The extracted DNA was phosphorylated at the 5′ end with T4 polynucleotide kinase (TaKaRa Bio, Shiga, Japan) and self-ligated with T4 DNA ligase (TaKaRa Bio). The rCTB–YVAD secretion vector pSCTB–YVAD was confirmed by sequencing, and then introduced into L. casei by electroporation, as described previously .
Secretion of rCTB–YVAD by L. casei
Overnight cultures of L. casei transformed with pSCTB–YVAD or pSCTB were inoculated into MRS/K medium to an optical density at 600 nm (OD600) of 0.05, and the cells were grown at 30°C until they reached an OD600 of 2.0. The culture supernatants were collected by centrifugation (7,000 × g, 5 min, 4°C) and concentrated 10-fold with Amicon Ultra Centrifugal Filter Units (10 kDa; Merck Millipore, Tokyo, Japan). The secretion and specific GM1-ganglioside-binding activities of rCTB–YVAD and rCTB in the concentrated supernatant were confirmed with immunoblotting using an antibody directed against CT or a GM1 enzyme-linked immunosorbent assay (GM1-ELISA), respectively.
The concentrated supernatants of L. casei (20 μl/lane), rCTB–YVAD (100 ng/lane), and cellular protein extracts of Caco-2 cells (50 μg/lane) were subjected to immunoblotting analysis. They were separated with SDS-PAGE (10–18%) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK). Antibodies directed against CT (Sigma-Aldrich, St. Louis, MO) and β-actin (Cell Signaling Technology, Boston, MA) were used as the primary antibodies. Alkaline phosphatase (AP)-labeled anti-rabbit IgG antibody (Cell Signaling Technology) was used as the secondary antibody, and binding was detected with a chemiluminescent substrate of AP (CDP-Star Reagent; Biolabs, Beverly, MA).
A GM1-ELISA was performed to determine specific GM1-ganglioside-binding activities. Briefly, 96-well microtiter plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were coated with 5 μg/ml monosialoganglioside GM1 (Sigma-Aldrich) diluted in bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), and incubated overnight at 4°C. After incubation, the plates were washed three times with PBS supplemented with 0.05% Tween-20 (PBS-T), and then blocked with PBS containing 1% bovine serum albumin (Nacalai Tesque, Kyoto, Japan) at 37°C for 2 h. After the plates were washed, the concentrated supernatant of L. casei (100 μl/well), rCTB–YVAD (50 ng/well), or rCTB (50 ng/well) was applied to the wells and incubated at 37°C for 2 h. The plates were incubated at 37°C for 2 h with antibody directed against CT and AP-labeled anti-rabbit IgG antibody used as the primary and secondary antibodies, respectively. The plates were incubated with AP substrate (Sigma-Aldrich) at 37°C for 20 min, and the OD405 was then measured with a microplate reader (ImmunoMini Nj-2300; Nunc, Rochester, NY).
Purification of rCTB–YVAD secreted by L. casei
rCTB–YVAD from the culture supernatant of L. casei transformed with pSCTB–YVAD was purified using the His-tag and an affinity resin containing bound nickel ions. The culture supernatant of L. casei transformed with pSCTB–YVAD was collected by centrifugation (12,000 × g, 30 min, 4°C) after growth in MRS/K medium at 30°C until the OD600 was 2.0. After the supernatant was filtered at 0.22 μm, imidazole was added to a final concentration of 20 mM, and the culture supernatant was then adjusted to pH 7.0. Nickel resin (Ni Sepharose High Performance; GE Healthcare) was added to the culture supernatant and then mixed gently overnight at 4°C. The open column was filled with resin, and then washed with wash buffer (10 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). rCTB–YVAD bound with nickel resin was eluted with elution buffer (10 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4). The eluted rCTB–YVAD was concentrated and the buffer replaced with PBS using Amicon Ultra Centrifugal Filter Units (10 kDa). The protein concentration of rCTB–YVAD was determined with Coomassie Protein Assay Reagent (Pierce, Perbio Science, Bonn, Germany). The purity of rCTB–YVAD was confirmed with SDS-PAGE on 17% polyacrylamide gel, followed by CBB staining and immunoblotting using an antibody directed against CT. The GM1-ganglioside-binding activities of purified rCTB–YVAD and rCTB were confirmed with GM1-ELISA.
Caco-2 cell viability assay
Caco-2 cells were cultured as described previously . Aliquots of 5 × 103 Caco-2 cells were plated in each well of a 96-well plate (Nunc). The cells were treated with three concentrations of rCTB–YVAD or rCTB (10, 20, or 50 μM) in the presence of 10 μg/ml LPS from E. coli O55:B5 (Sigma-Aldrich). After incubation for 48 h, 20 μl of WST-1 Cell Proliferation Reagent (TaKaRa Bio) was added to each well. After 2 h, the OD450 and OD630 were measured with a microplate reader. Cell viability was calculated as (OD450 – OD630 of treated cells/OD450 – OD630 of untreated control cells) × 100%.
Detection of translocated rCTB–YVAD and rCTB in Caco-2 cells
Aliquots of 7 × 105 Caco-2 cells were plated in each well of six-well plates (Nunc). Cells were treated with 50 μM rCTB–YVAD or rCTB in the absence or presence of 10 μg/ml LPS. After incubation for 6 h, cellular protein extracts were prepared with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Kyungki-Do, South Korea), according to the manufacturer’s protocol. The protein concentrations of the cellular protein extracts were determined with Coomassie Protein Assay Reagent. Intracellular rCTB–YVAD and rCTB were detected by immunoblotting with an antibody directed against CT. Equal loading was confirmed with an antibody directed against β-actin.
Inhibitory effect on caspase-1 activity
Caspase-1 activity was determined with a modification of a previously described method [20, 21]. Aliquots of 1 × 107 Caco-2 cells were plated in 90 mm plastic culture dishes (Nunc) and treated with or without 10 μg/ml LPS for 12 h. The cells were washed with PBS and resuspended in buffer W (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, pH 7.4) supplemented with 10 mM DTT and 1 mM phenylmethylsulfonyl fluoride. The cells were incubated at 4°C for 15 min and disrupted by 20 passages through a 23G needle. The lysates were then centrifuged at 12,000 × g for 5 min at 4°C and the supernatants collected. The protein concentrations of the lysates were determined with Coomassie Protein Assay Reagent. Aliquots of 10 μg/μl lysate were incubated at 30°C for 2 h in the absence or presence of 50 μM rCTB–YVAD or rCTB. The caspase-1 activity in the 10-fold-diluted lysate was determined with a Caspase 1 Assay Kit, Colorimetric (Calbiochem, La Jolla, CA).
Measurement of IL-1β by ELISA
Aliquots of 7 × 105 Caco-2 cells were plated in each well of six-well plates. The cells were treated with 50 μM rCTB–YVAD and rCTB in the presence of 10 μg/ml LPS for 48 h. The cell supernatants were centrifuged at 15,000 × g for 5 min at 4°C and stored at −80°C until IL-1β analysis. The concentrations of IL-1β in the cell supernatants were determined with a human IL-1β ELISA Kit (R&D Systems, Abingdon, UK).
Data are presented as means ± SEM. Statistical analyses were performed with Origin Pro 8.1 (OriginLab, Northampton, MA). Differences were analyzed with one-way ANOVA followed by Tukey’s test. In all analyses, P < 0.05 was deemed to indicate significance.