Characterization of Immunological Cross-Reactivity between Enterotoxigenic Escherichia coli Heat-Stable Toxin and Human Guanylin and Uroguanylin

Structural modeling and molecular dynamics simulations.Structural models of the full-length peptides of STh and STp were built by using MODELLER (38). The available experimental structures of the toxic domain of STp (PDB accession no. 1ETN) and the endogenous peptides guanylin (PDB accession no. 1GNA) and uroguanylin (PDB accession no. 1UYA) were used as templates. The structural model of STh and the solution structures of guanylin and uroguanylin were subjected to energy minimization by using Charmm (39), and subsequent molecular dynamics (MD) simulations were performed with the program NAMD2.9 (40, 41), using the Charmm27 force field (42, 43). The peptides were solvated in a cubic box (60 by 60 by 60 ų) of TIP3P water molecules by using Charmm GUI (44), which was also used to add sodium and chloride ions (0.15 M NaCl). The simulations were performed in the NPT ensemble at a temperature of 300 K and a pressure of 10⁵ Pa, with an integration time step of 1 fs. A cutoff of 14 Å was used for the truncation of nonbonded integration. A switch function was used for van der Waals forces, and a shift function was used for electrostatics. The simulations consisted of four successive heating phases (10 K, 100 K, 200 K, and 300 K), a 100-ps-long equilibration phase, and a production phase of 20 ns.

Peptides.STh and STp peptides used in this study either were purified from ETEC and kindly provided by John D. Clements, Tulane University, and Donald C. Robertson, Kansas State University, or had been produced by chemical synthesis (Bachem AG, Bubendorf, Switzerland). Synthetic STp (catalog no. H-6248) was synthesized with specific disulfide bridges (i.e., Cys5-Cys10, Cys6-Cys14, and Cys9-Cys17), while STh was a custom-made synthesized air-oxidized peptide. The guanylin (catalog no. H-2996) and uroguanylin (catalog no. H-2166) synthetic peptides with native disulfide bridges (Cys4-Cys12 and Cys7-Cys15 for both peptides) were also prepared by Bachem. The biological activity of all peptides was confirmed by a T-84 cell assay, and the observed 50% effective concentrations (EC₅₀s) were 58 ± 9 nM for ETEC STh, 44 ± 6 nM for ETEC STp, 79 ± 10 nM for synthetic STh, 161 ± 26 nM for synthetic STp, 692 ± 101 nM for guanylin, and 174 ± 21 nM for uroguanylin.

Antibodies.Polyclonal anti-STh and anti-STp antibodies, kindly provided by John D. Clements, Tulane University, were raised against bovine serum albumin (BSA) conjugates in rabbits and protein A purified. The conjugates were prepared by glutaraldehyde conjugation of STh and STp, respectively. Anti-STh MAb ST:G8, kindly provided by Sandhya S. Visweswariah, Indian Institute of Science, was described previously (37). This MAb was raised against a mutant variant of STh (SThY19F) conjugated to BSA by using glutaraldehyde. Three anti-STp MAbs (Fitzgerald Industries International), clone 29 (catalog no. 10-1013), clone 30 (catalog no. 10-1014), and clone 31 (catalog no. 10-1015), were all raised against a conjugate where synthetic STp with native disulfide bridges (catalog no. H-6248; Bachem) was conjugated to keyhole limpet hemocyanin (KLH). The conjugation was performed to selectively target carboxyl groups, thus coupling STp to KLH via the C-terminal carboxyl group or Glu7. Polyclonal antiguanylin (catalog no. ab14344; Abcam, Cambridge, United Kingdom) was produced in rabbit and raised against a synthetic peptide with the sequence PGTCEICAYAACTGC. According to the manufacturer, the peptide was conjugated to KLH by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The antiuroguanylin antibody (catalog no. sc-32578, lot no. J2010; Santa Cruz Biotechnology, Dallas, TX) was an affinity-purified polyclonal antibody produced in goat. The antibody was raised against a peptide that maps to the C terminus of human uroguanylin. Polyclonal antibodies used as negative controls were anti-GST rabbit polyclonal antibody (raised against glutathione S-transferase [GST]) (catalog no. sc-459; Santa Cruz Biotechnology) and anti-NATH rabbit polyclonal antibody (custom antibody raised against N-acetyltransferase, human; Biogenes GmbH, Berlin, Germany). The following secondary antibodies where purchased from Sigma-Aldrich (St. Louis, MO): anti-rabbit (IgG alkaline phosphatase conjugate A8025) for use in the STh ELISA, STp ELISA, and guanylin ELISA; anti-goat (IgG alkaline phosphatase conjugate A4187) for the uroguanylin ELISA; and anti-mouse (IgG alkaline phosphatase conjugate A4312) for all MAb ELISAs.

Conjugation procedure for ELISA coating.The conjugates used for ELISA coating in the STp ELISA, the STh ELISA, and the MAb ELISAs for ST:G8, clone 29, and clone 30 were prepared by glutaraldehyde conjugation of ST to ovalbumin according to a protocol modified from the one described previously by Lockwood and Robertson (11). The sources of ST were ETEC STp for the STp ELISA, ETEC STh for the STh ELISA, and synthetic STh for the MAb ELISAs. Briefly, 200 μg ST was conjugated to 450 μg ovalbumin (Sigma-Aldrich) by adding 600 μg glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 6.8), in a total reaction mixture volume of 1 ml. The reaction was allowed to proceed for 2 h with gentle stirring in the dark at room temperature. The reaction was stopped by the addition of 10 μl 1 M l-lysine (Sigma-Aldrich), and the mixture was stirred gently for another 30 min. The conjugate was then dialyzed against phosphate-buffered saline (PBS) for 24 h at 4°C by using Spectrapor dialysis tubing (molecular mass cutoff of 10 kDa) and stored at −20°C until use.

Mass spectrometry.Mass analysis was performed by using an Ultraflex MALDI ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA). Samples were desalted and prepared for analysis as described previously (45), using a matrix solution consisting of 8 g/liter alfa-cyano-4-hydroxycinnamic acid (CHCA), 60% acetonitrile, 15% methanol, and 0.1% trifluoroacetic acid (TFA). We also analyzed peptides after treatment with dithiothreitol (DTT) and iodoacetamide for reduction and alkylation of cysteines, as described previously (46). The mass spectrometer was calibrated prior to use with Peptide Calibration Standard II (Bruker Daltonics).

T-84 cell assay.T-84 cells (ATCC) were seeded and grown to confluence on 24-well plates (Nunc, Roskilde, Denmark) in Dulbecco's modified Eagle medium (DMEM)–F-12 medium (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum and 0.2% gentamicin. Cells were washed 3 times with 500 μl DMEM–F-12 medium and preincubated with 200 μl DMEM containing 1 mM 3-isobutyl-1-methylxanthine for 45 to 60 min at 37°C. Serial dilutions of peptides to be tested were diluted in 200 μl DMEM–F-12 medium, added to each well, and incubated for 60 min at 37°C. Samples were tested in duplicate wells. Following incubation, the reaction medium was aspirated, and the cells were lysed with 0.1 M HCl at 20°C for 20 min. In some instances, the cells were also freeze-thawed three times to ensure complete lysis. The lysates were centrifuged at 16,000 × g for 10 min, and supernatants were collected for analysis. Cyclic GMP (cGMP) levels were measured by using a commercial cGMP enzyme immunoassay kit (Enzo Life Sciences Inc.) according to the manufacturer's instructions.

T-84 cell assay neutralization experiments.For neutralization experiments, 10 ng STh was preincubated overnight at 4°C with polyclonal antibody diluted in 200 μl DMEM–F-12 medium. Antibody dilutions of 1:10, 1:50, and 1:100 were tested. For neutralization experiments with MAbs, the protocol was slightly modified: MAbs were diluted in PBS containing 0.2% bovine serum albumin and were preincubated with 10 ng ST for 1 h at room temperature prior to addition to T-84 cells. Neutralization experiments with MAbs were carried out with STh and STp. The T-84 cell assay was performed as described above to test the biological activity of the preincubated sample.

Competitive ST ELISAs.Competitive ST ELISAs using polyclonal anti-STh and anti-STp antibodies were performed as described previously (4, 11, 47), with minor modifications. Briefly, microtiter plates were coated with STh or STp ovalbumin conjugates (0.11 μg per well for STh and 0.16 μg per well for STp) in 150 μl ELISA buffer (128 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄ [pH 7.2]) overnight at 37°C. Wells were then washed and blocked with 1% ovalbumin in ELISA buffer for 1 h at 37°C. Seventy-five microliters of sample was mixed with 75 μl of diluted primary antibody (anti-STh, 1:2,000 final dilution; anti-STp, 1:10,000 final dilution) and incubated for 2 h at 37°C. After washing, the plate was incubated with 100 μl anti-rabbit secondary antibody (dilution, 1:400) for 1 h at room temperature. The plate was then developed for approximately 30 min by using 100 μl freshly prepared developing buffer (1 mg/ml para-nitrophenylphosphate (pNPP) in 9.7% [vol/vol] 0.5 mM MgCl₂ · 6H₂O [pH 9.8]), development was stopped by the addition of 50 μl 2 M NaOH, and the optical density at 405 nm (OD₄₀₅) was measured.

The competitive ELISAs using the ST:G8, clone 29, and clone 30 monoclonal antibodies were similar and differed only in coating and antibody concentrations. Briefly, ELISA plates (Nunc Immobilizer Amino) were coated with 100 μl of the STh-ovalbumin conjugate in PBS (146 mM NaCl, 4 mM Na₂HPO₄, 1.1 mM KH₂PO₄ [pH 7.2]) and incubated overnight at 4°C. For the ST:G8 ELISA, 0.020 μg conjugate per well was used; for clone 29 and clone 30, plates were coated with 0.005 μg conjugate per well. Subsequently, plates were blocked with 1% ovalbumin in PBS-T (PBS with 0.05% [vol/vol] Tween 20) for 1 h at 20°C with shaking. After washing with PBS-T, a volume of 60 μl of sample and 60 μl of primary antibody was added and incubated for 90 min at 20°C with shaking. Final antibody dilutions were 1:6,500 for ST:G8, 1:16,000 for clone 29, and 1:30,000 for clone 30. The plates were then washed with PBS-T and incubated at room temperature for 1 h with 100 μl anti-mouse secondary antibody diluted in PBS-T (ST:G8, 1:400; clones 29 and 30, 1:2,000). After a final wash, the plates were developed with 100 μl developing buffer for 15 to 20 min, development was stopped by the addition of 50 μl 2 M NaOH, and the OD₄₀₅ was measured.

The competitive ELISA using the anti-STp clone 31 MAb was performed by using a different coating approach. ELISA plates (Nunc CovaLink) were coated with synthetic STp according to the manufacturer's instructions. Briefly, 50 μl of 1 μM STp in PBS was mixed with 50 μl of an EDC solution [184 μg bis(sulfosuccinimidyl)suberate (BS3) and 50 mg EDC in PBS] and added to all wells, and the wells were incubated overnight at 20°C. The ELISA was then performed with washing steps, blocking steps, and development as described above. The final concentration of clone 31 primary antibody was 1:16,000, and that of anti-mouse secondary antibody was 1:500.

The ETEC and synthetic STh and STp peptides had similar antigenic properties in all ELISAs, and for each ELISA, at least one experiment was performed by using ETEC STh and STp.

Cloning and expression of proguanylin and prouroguanylin.The proforms of guanylin and uroguanylin were chosen as coatings for guanylin and uroguanylin competitive ELISAs to ensure binding and competition of A-form-specific antibodies (48). The cDNA sequences of proguanylin (IMAGE ID 100062076) and prouroguanylin (IMAGE ID 7939624; http://www.imageconsortium.org) were cloned into the pSXG vector (49) as GST fusions. Standard protocols were used (50). The two sequences were amplified by PCR with the mutagenic primers listed in Table 1. This introduced EcoRI and BamHI restriction sites on each end of the PCR products. The PCR products and the empty pSXG vector were subsequently digested with EcoRI and BamHI, and these were ligated, resulting in in-frame fusions of the GST coding sequence (CDS) and the proguanylin and prouroguanylin CDSs, respectively. The resulting plasmid carrying the proguanylin sequence was named pSXG-ProG-GN, and the one carrying the prouroguanylin sequence was named pSXG-ProU-UGN. Due to the expression of insoluble protein from plasmid pSXG-ProU-UGN, an additional plasmid, pSXG-ProG-UGN, was constructed by replacing the mature peptide-encoding sequence of guanylin in the pSXG-ProG-GN vector with the peptide-encoding sequence of uroguanylin. Plasmid pSXG-ProU-UGN was used as the template in a PCR using the primers listed in Table 1. The resulting PCR product contained the sequence of the uroguanylin peptide and a part of the pSXG vector flanked by the restriction sites BbvCI and AatII. The PCR product and the pSXG-ProG-GN vector were then both digested with BbvCI and AatII and subsequently ligated. All plasmids were verified by sequencing.

The GST-proguanylin-guanylin (GST-ProG-GN) and GST-proguanylin-uroguanylin (GST-ProG-UGN) fusion proteins were expressed from plasmids pSXG-ProG-GN and pSXG-ProG-UGN, respectively, using E. coli Origami B cells (Novagen/Merck, Darmstadt, Germany). Transformations and inoculations were performed according to the manufacturer's instructions, and the bacteria were grown in 500-ml cultures of LB medium supplemented with appropriate antibiotics at 37°C. At an OD₆₀₀ of 0.5, protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 400 μM. The cultures were incubated for an additional 3 to 4 h at 37°C before they were harvested by centrifugation at 17,000 × g for 10 min at 4°C. The pellets were resuspended in 10 ml PBS buffer with 200 mM NaCl and lysed by sonication. Samples were subsequently centrifuged in Corex tubes at 48,000 × g for 20 min at 4°C, and supernatants were transferred into clean 15-ml tubes. The GST-tagged proteins were purified by using 1 ml of a 50% glutathione-Sepharose 4B slurry (GE Healthcare, Little Chalfont, United Kingdom) per sample, according to the manufacturer's instructions. The elution step was repeated three times. Purified fusion proteins were examined by SDS-PAGE using 12% slab gels. Both pSXG-ProG-GN and pSXG-ProG-UGN showed a single prominent band at a molecular mass of 38 kDa, and Western blots with antiguanylin and antiuroguanylin antibodies confirmed the identities of the fusion proteins. Protein concentrations were estimated by measuring the absorbance at 280 nm.

Competitive guanylin and uroguanylin ELISAs.Competitive guanylin and uroguanylin ELISAs were developed based on a standard protocol from the antibody supplier (Abcam), and coating steps were performed according to the plate manufacturer's instructions. ELISA plates (Nunc Immobilizer glutathione) were coated with 100 μl GST-tagged fusion proteins (50 nM GST-ProG-UGN and 25 nM GST-ProG-GN) per well in PBS (104.7 mM NaCl, 10.6 mM Na₂HPO₄, 2.9 mM KH₂PO₄ [pH 7.2]) and incubated overnight at 4°C. The plates were subsequently washed and blocked for 1 h with PBS-T at 20°C with shaking. After washing, 55 μl of sample was mixed with 55 μl of primary antibody solution (final dilutions in PBS-T of 1:1,000 for antiuroguanylin and 1:4,000 for antiguanylin), added to each well, and incubated for 90 min at 20°C with shaking. After washing, 100 μl secondary antibody diluted in PBS-T (final dilution 1:400 of anti-goat for antiuroguanylin and 1:400 of anti-rabbit for antiguanylin) was added per well, followed by incubation at 20°C for 1 h. After washing, the plates were developed with 100 μl developing buffer for 30 to 40 min, development was stopped by the addition of 50 μl 2 M NaOH, and the OD₄₀₅ was read.

Statistical and regression analyses.The R statistical computing environment was used for statistical and regression analyses (http://www.r-project.org/). Analysis of variance (ANOVA) and Tukey's honestly significant difference test were used to evaluate whether the abilities of the individual peptides to inhibit binding to ELISA coatings in the polyclonal ELISAs were significantly different (P < 0.05). To calculate the half-maximal inhibitory concentration (IC₅₀) in the monoclonal antibody ST:G8 ELISA and the EC₅₀ in the T-84 cell assay, four-parameter log-logistic regression was performed by using the drc R package (51). For IC₅₀ calculations, all peptides were assumed to have the same minimum, and for EC₅₀ calculations, the maximum values and slopes were also assumed to be the same. Relative IC₅₀s and their significances were calculated with the drc selectivity index (SI) function.