Bacterial strains, plasmids and growth conditions
E. coli DH5α (Invitrogen, Carlsbad, CA) was grown in LB media at 37°C with 220 rpm orbital shaking or on LB-agar plates at 37°C. Lactobacilli were grown in lactobacilli MRS broth (Difco, Sparks, MD) at 37°C without agitation or anaerobically on MRS-agar plates (BD - GazPak EZ, Sparks, MD). Antibiotics were added at the following concentrations when indicated: ampicilin (100 μg/ml) and erythromycin (300 μg/ml E. coli and 5 μg/ml lactobacilli).
Construction of recombinant Lactobacillus and E. colistrains
The 1H scFv was amplified from the pMoPac16 vector containing the 1H scFv  using the primers; anthrx1H-Fw: 5''-CCGGCCATGGATGATATTCAGATGACACAGACTAC-3'' and anthrx1H-Rv: 5''-GCACCTGCGGCCGCCGAGGAGACGGTGACTGAG-3''. The PCR fragment was cloned into pGEM®-T easy vector (Promega, Madison, WI) and DNA sequence verified by sequencing. The 1H scFv gene was excised using NcoI and NotI restriction enzymes (Promega) and ligated into the NcoI/NotI digested Lactobacillus expression vectors, pAF100, pAF400 and pAF900  giving plasmids pAF100-1HscFv, pAF400-1HscFv and pAF900-1HscFv for secreted, attached and anchored expression respectively. The expression plasmids were transformed into L. paracasei (previously known as L. casei or L. zeae ATCC 393 pLZ15- ) by electroporation as previously described [23, 33], generating the Lactobacillus strains KKA307, KKA308 and KKA317 expressing the 1H scFv anchored, secreted and attached respectively. A Lactobacillus strain, KKA101, harboring a non-expressing version of the plasmid was constructed by transforming L. paracasei with the empty pIAV7 plasmid . The Lactobacillus strain L. paracasei pAF400, expressing an attached scFv against an irrelevant antigen (SAI/II from S. mutans) has been described previously .
An E. coli strain for periplasmic expression of the 1H scFv was constructed by amplifying the scFv fused to the E-tag from pAF900-1HscFv with primers anthrx1H-pOPE-Fw: CGGCCATGGCGGATATTCAGATGACACAGACTAC and pOPE-Etag-Rv: CCGTATCCGGACCCGCTGGAACCGCGTCATCATCACCATCATCATTAA TCTAGAGCC. The PCR fragment was restriction digested with NcoI and BglII (Promega) and cloned into the NcoI/BglII digested plasmid pOPE101-215(yol)  generating pOPE101-1HscFv(E-tag). The plasmid was transformed into E. coli XL1-Blue competent cells (Agilent, Santa Clara, CA) by electroporation generating the strain KKA300 and the DNA sequence verified by sequencing.
The transformants were grown in MRS with 5 μg/ml erythromycin until an OD600 of 1.0. The cultures were centrifuged at 3,200 × g to separate the pellet from the supernatant. The supernatant was filter sterilised, pH adjusted to 7.0, dialysed against 10 mM Tris (pH 8.0) and concentrated using Amicon Ultra-4 centrifugal filter units (10 kDa cut off, Millipore, Carrigtwohill, Co. Cork, Ireland). The concentrated supernatant was mixed with 2 × Laemmli buffer and boiled for 5 minutes (min). The cell culture pellet was washed twice with PBS, resuspended in 100 μl Laemmli buffer and boiled for 5 min. The cell extract was centrifuged at 16,000 × g to remove cell debris and the supernatant containing soluble proteins was kept. The supernatant and cell extract were run on a 10% SDS-polyacrylamide gel at 170 volts and the proteins were transferred onto a nitrocellulose membrane (Hybond-ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membrane was blocked with PBS-T (PBS with 0.05% (v/v) Tween 20 + 5% (w/v) milk powder) and successively incubated with mouse anti-E-tag antibodies (1 μg/ml, GE-Healthcare) and HRP (horse radish peroxidase) labelled goat anti-mouse antibodies (DAKO A/S, Glostrup Denmark). The signal was detected by chemiluminescence using the ECL Plus™ Western Blotting detection system (GE Healthcare).
For re-attachment of 1H scFv on lactobacilli, strains KKA317 and KKA101 were grown in 50 ml MRS with 5 μg/ml erythromycin until OD600 of 1.0. Cultures were harvested by centrifugation and supernatant filter sterilised and adjusted to pH 7.2. The conditioned media were re-inoculated with KKA317 and KKA101 at an OD600 of 0.2 and grown to OD600 of 1.0. Cell pellets were treated as previously described and run on an 10% SDS-polyacrylamide gel and Western blotted.
Enzyme-Linked ImmunoSorbent Assay (ELISA)
96 well microtiter plates (EIR/RIA plate, Costar, Lowell, MA) were coated with 100 μl rPA (List labs, Campbell, CA) at 1 μg/ml in PBS overnight (o/n) at 4°C. Plates were subsequently blocked with 200 μl 1% BSA (in PBS containing 0.05% Tween 20, PBS-T) for two hours at 4°C. After washing with PBS-T, dilutions of scFv producing Lactobacillus culture supernatants were added and the plates incubated at 4°C o/n. ScFvs purified from E. coli were used as a positive control for quantification. Plates were subsequently washed three times and 100 μl mouse anti-E-tag antibody (GE-healthcare) was added (1 μg/ml) in blocking solution, followed by incubation at room temperature for 2 h. Plates were then washed three times in PBS-T and incubated with 100 μl AP conjugated rabbit anti-mouse antibody at 1/1000 (Dako A/S, Glostrup Denmark) in blocking solution. Following an additional 1 hour incubation at room temperature, the plates were washed twice in PBS-T and once in PBS, resuspended in 100 μl of diethanolamine buffer (1M, pH 10.0) containing 1 mg/ml pNPP (Sigma-Aldrich, St. Louis, MO) and absorbance was read after 10-30 min at 405 nm in a Varioskan Flash (Thermo Scientific, Waltham, MA).
50 μl of Lactobacillus cultures grown to an OD600 of 1.0 in MRS were harvested by centrifugation (8000 rpm, 1 min) and washed three times in PBS. Bacteria were resuspended in 50 μl PBS with 1% BSA (PBS-BSA) and incubated for 30 min on ice sequentially with 30 μl PA coated beads (Invitrogen), 50 μl anti-E-tag antibody (10 μg/ml) and 50 μl FITC conjugated anti-mouse immunoglobulins (diluted 1/100) (Jackson Immunoresearch Laboratories, West Growe, PA), all diluted in PBS-BSA. Bacteria were washed with 500 μl PBS between all three incubations. Samples were resuspended and fixed in 300 μl 2% paraformaldehyde in PBS and analysed using a FACS Calibur machine (Becton Dickinson, Franklin Lakes, NJ). 1 μm red FluoSpheres®microspheres (Invitrogen) were incubated with rPA (List labs) according to manufactors instructions to generate PA coated fluorescent beads for use in flow cytometry.
Macrophage toxicity assay to assess neutralisation by scFvs
Protection by Lactobacillus and E. coli produced scFvs were analysed by their capacity to protect the J774 MΦ cell line from killing by LT [36, 37]. Briefly, J774 MΦ were added to 96-well, flat-bottom wells (5 × 104 MΦ/well) and incubated at 37°C in 5% CO2 in air. After 12 hours of incubation, LT (i.e., 1 μg/ml rPA and 1 μg/ml LF, (List labs)) pre-mixed with scFvs were added to the cultures and incubated for an additional 12 hours. Viable MΦ were evaluated by colorimetric assay by reading absorption at 562nm after addition of Methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich) . MTT was used at a concentration of 5 mg/ml, and a volume of 20 μl (100 μg/well) was added to individual wells.
Purification of scFvs
ScFv was purified from the supernatant of strains KKA308 and KKA317, grown in defined minimal media . The scFv was isolated on a HiTrap™ anti-E-Tag Column (GE-healthcare) according to the manufactures instructions. Eluate was concentrated on Amicon Ultra-4 10K MWCO spin column (Millipore, Billerica, MA). The concentration of purified scFv was determined using the Micro BCA™ Protein Assay kit (Pierce, Rockford, IL) with BSA as a standard.
The 1H scFv was isolated from the periplasma of the recombinant E. coli strain, KKA300, as previously described  with the following modifications. The culture was grown in 500 ml YT-broth supplemented with 100 mM glucose and 100 μg/ml ampicilin. The periplasmic extract dialyzed against PBS was adjusted to 30 mM imidazole (Sigma Aldrich) and 0.5 M NaCl (pH 7.5). The adjusted periplasmic extract was immobilised on a 5 ml HisTrap™ HP Column (GE-healthcare) and washed with 20 bed volumes wash buffer (PBS, 30 mM imidazole, 0.5 M NaCl, pH 7.5) and subsequently eluted with 5 bed volumes elution buffer (PBS, 0.5 M imidazole, 0.5 M NaCl, pH 7.5). Eluate was concentrated and buffer exchanged with PBS on a Amicon Ultra-4 10K MWCO spin column (Millipore, Billerica, MA) and purified scFv concentration determined as described above.
Female C57BL/6 mice, six-seven weeks of age, were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions and provided food and water ad libitum. All studies were performed in accordance with both National Institutes of Health and Institutional guidelines and approved by the Ohio State University Institutional Animal Care and Use Committee (Protocol number 2009A0210).
A dose study of the oral effect of ET was carried out on groups of mice challenged with 10, 25, 50 and 100 μg of ET (equal amount of rPA plus EF (List Labs)) given in 100 μl PBS by gavage. After 16 hours, the toxic effect was measured as ET induced fluid accumulation in the small and large intestine. Mice were euthanized with CO2 and death confirmed by cervical dislocation prior to removal of small and large intestine. Fluid accumulation was measured as percent of the weight of the small and large intestine compared to total body weight.
The KKA307, KKA308, KKA317 and L. paracasei pAF400  strains were grown in MRS to an OD600 of 1.0, harvested by centrifugation and resuspended in culture supernatant with pH adjusted to 7.0 to give 5 × 109 cfu/ml. Nine-twelve weeks old C57BL/6 mice (body weight 15-20 g) were given 2.5 × 109 cfu recombinant Lactobacillus by gavage. Four hours later they were challenged with a non-lethal dose of 50 μg ET (50 μg rPA plus 50 μg EF (List Labs)) together with an additional 2.5 × 109 cfu recombinant Lactobacillus by gavage. After 16 hours, the toxic effect of the ET was measured as fluid accumulation in the small and large intestine.
The relative intestinal weight, in percent of total body weight of the treated groups, were compared to mice group receiving ET only, and analysed with the Mann-Whitney U-test using the GraphPad Prism software.