Fibronectin Binding to the Salmonella enterica Serotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat

The expression and purification of a glutathione S-transferase (GST)-ShdA fusion protein containing residues 59 to 1553 of ShdA (G-S_59-1553) have been described previously (22). Plasmids for expression of GST fusion proteins with smaller segments of ShdA (Fig. 1) were constructed by PCR amplification of the corresponding regions in the shdA gene and subsequent cloning into the pGEX4T/1 vector restriction enzymes EcoRI and SalI (Amersham Pharmacia). For expression of fusion proteins, we used E. coli strain BL21 (Invitrogen). This strain, containing the recombinant plasmids, was grown in Luria-Bertani broth supplemented with 100 mg of carbenicillin/liter to mid-log phase at 37°C with shaking, and expression was induced by the addition of IPTG (isopropyl-β-d-thiogalactopyranoside) (Sigma, St. Louis, Mo.) to a final concentration of 1 mM. After incubation at 37°C with shaking for 4 h, the cells were harvested by centrifugation at 10,000 × g for 20 min. The pellet was resuspended in 15 ml of phosphate-buffered saline (PBS), pH 7.4, and 5 ml of protease inhibitor cocktail (Sigma) was added. The cells were lysed using a French pressure cell at 12,000 lb/in², and the insoluble fraction was separated by centrifugation. Fusion proteins were purified from the soluble fraction by affinity chromatography using glutathione Sepharose (Amersham Pharmacia) according to the manufacturer's instructions. The fusion proteins were eluted with 10 mM reduced glutathione (Sigma) in 50 mM Tris-HCl, pH 8.5.

Binding of GST-ShdA fusion proteins to fibronectin-coated wells was determined using an enzyme-linked immunosorbent assay (ELISA). Wells of a 96-well plate (Maxisorp; Nunc, Roskilde, Denmark) were coated with 50 μl of bovine fibronectin in 100 mM Tris-HCl, pH 8, per well for 12 h at 4°C at the concentrations indicated below. Additional binding sites were blocked by incubation of 200 μl of block buffer (2% [wt/vol] nonfat milk, 0.02% [vol/vol] Tween 20 in PBS, pH 7.4) per well for 1 h. The block buffer was removed, and the wells were washed three times with distilled H₂O. The appropriate amount of test ligand in a volume of 50 μl of PBS, pH 7.4, was added in the presence or absence of inhibitor, and the plate was incubated for 90 min at room temperature. The wells were washed three times with distilled H₂O. Bound ligand (G-S_59-1553, G-S_59-480, G-S_480-581, G-S_602-1048, G-S_1086-1553, or G-S_470-1553) was detected using a 1/1,000 dilution of goat anti-GST antiserum (Amersham Pharmacia) as the primary antibody and a 1/5,000 dilution of monoclonal anti-goat immunoglobulin G alkaline phosphatase conjugate as a secondary antibody. Alkaline phosphatase activity associated with each well was detected by the addition of 1 mg of p-nitrophenyl phosphate/ml in 100 mM glycine, 2 mM MgCl₂, and 1 mM ZnCl₂. Alternatively, biotinylated protein ligand was detected with 1/1,000 streptavidin-horseradish peroxidase polymer conjugate (Sigma) in PBS, pH 7.4, and 0.04% Tween 20. The horseradish peroxidase activity in each well was detected by the addition of 50 μl of 0.4-mg/ml o-phenylenediamine dihydrochloride- 0.4-mg/ml urea hydrogen peroxide in 50 mM phosphate citrate buffer. Protein was biotinylated using sulfo-succinimidyl-6-(biotinamido) hexanoate (Pierce, Rockford, Ill.) according to the manufacturer's instructions. Briefly, a 20-fold molar excess of sulfo-succinimidyl-6-(biotinamido) hexanoate was incubated with a 1.5-mg/ml solution of G-S_59-1553 or G-S_470-1553 in PBS, pH 7.4, at room temperature for 30 min. The remaining unreacted sulfo-succinimidyl-6-(biotinamido) hexanoate was removed by dialysis against 1 liter of PBS, pH 7.4, for 10 h at 4°C. The biotinylated protein was stored at −20°C for 1 month.

Antiserum was raised in 6-lb female New Zealand White rabbits. A prebleed (5 ml) was taken, followed by the primary immunization with a 1:1 mixture of fusion protein (0.5 mg) and Titer-Max Gold (Sigma) adjuvant (total volume, 1 ml) injected subcutaneously at >10 different locations. This procedure was repeated after 14 and 28 days. After a further 2 weeks, the rabbits were anesthetized with a Ketamine, Xylaxine, and Acepromazine cocktail and exsanguinated by cardiac puncture. The serum was prepared by standard methodology and preadsorbed with GST-glutathione Sepharose five times.

Production of monoclonal antibody (MAb) specific to G-S_470-1553 was performed essentially as described previously (3). For primary screening of monoclonal hybridoma supernatants, ELISA plates were coated overnight with 5 μg of G-S_470-1553 Tris-HCl (pH 8)/ml, and upon incubation of the hybridoma supernatants, mouse anti-ShdA was detected with a peroxidase-conjugated secondary antibody and H₂O₂-tetramethylbenzidine as the substrate system. Immunopositive culture wells were then submitted to a secondary screening to test for the MAb's capacity to block interaction of G-S_470-1553 with fibronectin. To this end, ELISA plates were coated with fibronectin (5 μg of Tris-HCl [pH 8]/ml). The plates were washed with distilled H₂O and then incubated with a mixture of the MAb and purified G-S_470-1553. Binding of G-S_470-1553 to fibronectin-coated wells was detected with polyclonal rabbit anti-ShdA (α-ShdA) antiserum and a peroxidase-conjugated secondary antibody as described above. The hybridomas that had lower photometric signals in this secondary screening than in a control binding assay containing no MAb were expanded, cloned by limiting dilution, and stored in liquid nitrogen for further use. Two MAbs that produced the lowest photometric signals in the binding assay were designated 3-9D 9F and 4-10A 11B, and a third that had no detectable inhibitory activity was designated 4-10G 8G. For mass production of MAb 3-9D 9F, we used a membrane-based high-density cell culture technology (CELLine 1000; Integra Biosciences, Ijamsville, Md.).

Binding of the passenger domain of ShdA (G-S_59-1553) (Fig. 1) to fibronectin-coated wells was described previously using ELISA (22). G-S_59-1553 had a predicted molecular mass of 178 kDa but migrated with an apparent molecular mass of >250 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A discrepancy between the predicted molecular mass and the apparent molecular mass is common for proteins containing acidic repeated sequences, including FnbpA, a fibronectin-binding protein of Staphylococcus aureus (26). The predicted passenger domain of ShdA is also highly acidic, containing 188 (∼12%) acidic residues (glutamate and aspartate) out of 1,498 total residues. To further characterize fibronectin-binding activity, we first determined the time dependency of binding using an ELISA. G-S_59-1553 was added to 96-well plates coated with 1 nmol of bovine fibronectin. After incubation for different time intervals, the wells were washed, and the amount of well-associated G-S_59-1553 was detected with α-GST antiserum using ELISA. The amount of bound G-S_59-1553 increased in a time-dependent manner for the first 60 min. Incubation of fibronectin and G-S_59-1553 for >60 min did not increase the amount of G-S_59-1553 bound to wells, indicating that binding equilibrium had been reached (data not shown). In all subsequent binding assays, we therefore incubated test peptides in fibronectin-coated wells for 60 min.

We next determined the concentration required for half-maximal binding at equilibrium of G-S_59-1553 to bovine plasma fibronectin by using an ELISA. Increasing concentrations of G-S_59-1553 were added to 96-well plates coated with 0.25 μg of bovine plasma fibronectin and incubated for 60 min. The amount of well-associated G-S_59-1553 was detected with α-GST antiserum using ELISA (Fig. 2A). Saturation of binding was reached at a G-S_59-1553 concentration of ∼3 × 10⁻⁷ M, and half-maximal binding occurred when the fusion protein was added to wells at a concentration of ∼1.2 × 10⁻⁷ M.

The ShdA passenger domain (residues 59 to 1553) of serotype Typhimurium consists of two regions, an N-terminal region (residues 59 to 470; nonrepeat region) followed by a second region (residues 470 to 1553; repeat region) composed almost entirely of two types of imperfect and direct amino acid repeats that we have designated repeat type A and repeat type B (Fig. 1). The type A repeat consists of an ∼100-amino-acid motif that is repeated three times. The type B repeat consists of a 60-amino-acid motif that is repeated nine times (23). To investigate whether either of these regions exhibits binding in the absence of the other, we constructed two GST fusion proteins consisting of residues 59 to 480 (G-S_59-480) and 470 to 1553 (G-S_470-1553) (Fig. 1). While G-S_59-480 migrates in SDS-PAGE at approximately its predicted molecular mass of 70 kDa, G-S_470-1553 migrated at ∼230 kDa, considerably higher than its predicted molecular mass of 136 kDa. This is likely due to the presence of acidic repeats in this region. The fibronectin-binding properties of these fusion proteins were compared by ELISA to those of GST and a GST fusion containing the entire ShdA passenger domain (G-S_59-1553) (Fig. 3A). Binding of the nonrepeat region (G-S_59-480) to fibronectin-coated wells was greater than that of GST but less than that of the entire ShdA passenger domain (G-S_59-1553). In contrast, the repeat region (G-S_470-1553) bound to immobilized fibronectin at a level comparable to that of the ShdA passenger domain (G-S_59-1553) under these conditions.

To determine the specificity of fibronectin binding by ShdA, we tested the abilities of GST and GST fusion proteins containing the nonrepeat region (G-S_59-480) or the repeat region (G-S_470-1553) to inhibit binding of a biotinylated GST fusion containing the ShdA passenger domain (biotin-G-S_59-1553) to bovine fibronectin in a solid-phase binding assay (Fig. 4A). A biotinylated GST fusion to the ShdA passenger domain (biotin-G-S_59-1553) (33 nM) was incubated in 96-well plates coated with bovine plasma fibronectin in the presence of increasing concentrations (0.6, 6, 60, and 600 nM) of either GST, G-S_59-1553, G-S_59-480, or G-S_470-1553. The GST fusion protein containing the ShdA passenger domain (G-S_59-1553) inhibited binding of biotin- G-S_59-1553 to fibronectin at a concentration of 6 nM and inhibited binding by ∼50% at 60 nM. The GST fusion protein containing the repeat region (G-S_470-1553) inhibited binding at a concentration of ≥60 nM, and the GST fusion containing the nonrepeat region (G-S_59-480) exhibited weak inhibition at a concentration of 600 nM (Fig. 4A). No inhibitory activity of GST was detectable up to 600 nM.

To estimate the affinity of fibronectin binding to the ShdA repeat region, we determined by ELISA the concentration required for half-maximum binding at equilibrium for fibronectin binding of G-S_470-1553. Increasing concentrations of the GST fusion protein containing the repeat region (G-S_470-1553) were added to 96-well plates coated with 5 μg of bovine plasma fibronectin/ml, and binding was allowed to proceed for 60 min (Fig. 2B). The concentration of G-S_470-1553 at which half-maximum binding to fibronectin was observed was ∼0.75 × 10⁻⁷ M. Thus, the ShdA passenger domain (Fig. 2A) and the ShdA repeat region (Fig. 2B) bound fibronectin with similar affinities. Together, these data suggested that the primary fibronectin-binding domain was contained within the repeat region (residues 470 to 1553) of the ShdA passenger domain.

To further investigate binding of the ShdA repeat region, three additional GST-tagged fusion proteins were constructed that together contained all of the repeats present in the ShdA passenger domain. The first contained repeat A1 (G-S_480-581), the second contained repeats B1 through B7 (G-S_602-1048), and the third contained repeats A2, B8, A3, and B9 (G-S_1086-1553) (Fig. 1). Both G-S_602-1048 and G-S_1086-1553 migrated at apparent molecular masses greater than their predicted molecular masses of 71 and 76 kDa, respectively, probably due to the presence of acidic repeats. None of these fusion proteins (G-S_480-581, G-S_602-1048, or G-S_1086-1553) bound to fibronectin-coated wells at levels comparable to that of the entire repeat region (G-S_470-1553) (Fig. 3B). While the GST fusion protein containing the A1 repeat (G-S_480-581) exhibited a small but significant increase in binding to fibronectin-coated wells compared to the GST control, fusion proteins G-S_602-1048 and G-S_1086-1553 did not exhibit greater binding than GST.

In order to address the question of whether the truncated proteins were able to cooperate to bind bovine fibronectin, we tested the ability of the A1 repeat (G-S_480-581), the B1 to B7 repeats (G-S_602-1048), and the A2, B8, A3, and B9 repeats (G-S_1086-1553) to inhibit the binding of a biotinylated GST fusion containing the repeat region of the ShdA passenger domain (biotin- G-S_59-1553) to bovine fibronectin in a solid-phase binding assay (Fig. 4B) individually and in combination. The biotinylated GST fusion to the repeat region (biotin- G-S_470-1553) was incubated in 96-well plates coated with bovine plasma in the presence of increasing concentrations of G-S_470-1553, G-S_480-581, G-S_602-1048, G-S_1086-1553, or GST. The GST fusion protein containing the complete repeat region (G-S_470-1553) inhibited binding of biotin- G-S_470-1553 to fibronectin at a concentration of 60 nM. The GST fusion proteins G-S_480-581, G-S_602-1048, and G-S_1086-1553 and GST, individually or in combination, did not exhibit inhibitory activity (Fig. 4B). This suggested that either the fibronectin-binding sites within the repeat region overlap the truncated recombinant proteins (G-S_480-581, G-S_602-1048, and G-S_1086-1553) and cannot function separately or the truncated proteins do not fold correctly.

A potential limitation of measuring the binding and/or inhibitory activities of truncated peptides derived from the ShdA passenger domain is that binding activity may be affected as a result of misfolding. To overcome these potential limitations, we determined the ability of rabbit immune sera specific for the truncated peptides of ShdA to inhibit the binding of the GST fusion protein containing the ShdA passenger domain (G-S_59-1553) to bovine fibronectin in a solid-phase binding assay. As a negative control, we constructed a GST fusion containing a region of the C-terminal domain of ShdA (residues 1594 to 1860), a region thought to mediate transport of the ShdA passenger domain across the bacterial outer membrane (G-S_1594-1860) (Fig. 1) but not fibronectin binding. Rabbit immune sera were raised against the GST fusion proteins containing the ShdA passenger domain (G-S_59-1553), repeat A1 (G-S_480-581), repeats B1 to B7 (G-S_602-1048), the region containing repeats A2, B8, A3, and B9 (G-S_1086-1553), and the C-terminal domain (G-S_1594-1860). To remove anti-GST antibodies, each serum was preadsorbed with GST bound to glutathione Sepharose resin. Following preadsorption, anti-GST antibodies were not detectable with any of the sera by Western blotting (Fig. 5). Antiserum raised against the ShdA passenger domain (α-G-S_59-1553) detected GST fusion proteins containing all regions of the ShdA passenger domain but did not detect the GST fusion protein raised against the C-terminal domain (G-S_1594-1860). Antiserum raised against the C-terminal domain fragment (G-S_1594-1860) detected only this protein. Antisera raised against G-S_480-581, G-S_602-1048, and G-S_1086-1553 each detected the passenger domain and the repeat region in addition to the protein used to immunize in each case. The abilities of these antisera to inhibit fibronectin binding by the ShdA passenger domain (G-S_59-1553) were determined by ELISA (Fig. 6). In the presence of antiserum raised against the ShdA passenger domain (α-G-S_59-1553), binding to fibronectin-coated 96-well plates was reduced to <10% of that observed in the presence of preimmune serum (Fig. 6). This indicated that antibodies in the polyclonal serum that recognized one or more epitopes of the ShdA passenger domain were able to compete with fibronectin for binding to ShdA. To further localize the positions of these epitopes, we tested the abilities of sera raised to the truncated peptides of the ShdA passenger domain to inhibit binding of the ShdA passenger domain to bovine fibronectin. Antisera raised against repeat A, repeats B1 to B7, or the C-terminal domain (α-G-S_1594-1860) did not significantly decrease binding of the ShdA passenger domain (G-S_59-1553) to bovine fibronectin. In contrast, the presence of antiserum raised to repeats A2, B8, A3, and B9 reduced binding to ∼40% of that in the presence of preimmune serum, suggesting that an important component of the ligand-binding domain may be contained in this region.

A second approach, complementary to that described in the previous section, was used to identify the region of ShdA that contained the fibronectin-binding domain. MAbs specific to ShdA were tested by ELISA for the ability to inhibit ShdA interaction with fibronectin-coated wells. Hybridoma cells were generated from suspensions of splenocytes from mice hyperimmunized with a recombinant protein containing residues 470 to 1553 of ShdA by fusion with Sp2/0 myeloma cells. Culture supernatants from 18 independent hybridoma cell lines that secreted immunoglobulin G specific for G-S_470-1553 varied in their abilities to inhibit ShdA binding to fibronectin-coated wells in an ELISA (data not shown). Three MAbs, designated 4-10A 11B, 3-9D 9F, and 4-10G 8G, were characterized further. Coincubation of MAbs 4-10A 11B and 3-9D 9F resulted in reduction of ShdA binding to fibronectin-coated wells to ∼6 and 25%, respectively, of binding in the absence of MAb. In contrast, coincubation of MAb 4-10G 8G did not result in reduced binding of ShdA to fibronectin-coated wells (Fig. 7). The locations of epitopes recognized by each MAb were identified by expression of recombinant N-terminal GST fusion proteins containing fragments of the ShdA repeat region in E. coli and subsequent separation by SDS-PAGE. Following Western transfer, the immobilized proteins were detected with each MAb or α-GST polyclonal serum (Fig. 8A). 4-10G 8G, which did not inhibit ShdA binding, recognized an epitope in the recombinant protein containing repeats B1 to B7 and one containing repeats A2, B8, A3, and B9 but not one containing repeat A1. In contrast, 4-10A 11B and 3-9D 9F recognized an epitope present in the recombinant protein containing repeats A2, B8, A3, and B9 but not that present in repeat A1 or repeats B1 to B7 (Fig. 8A). Further analysis with recombinant proteins containing smaller fragments from the region containing repeats A2, B8, A3, and B9 indicated that 4-10A 11B and 3-9D 9F recognize distinct epitopes (Fig. 8B). 4-10A 11B recognized an epitope in the recombinant protein containing repeats A2, B8, and A3, but smaller fragments, including ones with overlapping sequence, were not recognized. In contrast, 3-9D 9F recognized all recombinant proteins containing repeat A3. Repeat A3 is a 97-residue sequence containing 16 acidic residues.