The soluble extracellular domain of CD6 (sCD6) was produced as previously described (20), and the remaining recombinant proteins (N-SSc5D, Spα, and sCD5) were expressed and purified as follows. A cDNA corresponding to the N-terminal half of SSc5D (exons 1–12), which includes the five SRCR domains (N-SSc5D) (12), was amplified by PCR from human placenta cDNA using forward 5′-TATAATGGATCCGAGCGCCTGCGCCTGGCCGAT and reverse 5′-AATAGGATCCCTCTTGTGTCCGGCAGGCGCCTTATTGCTGG primers (BamHI restriction sites are underlined). The Spα cDNA was amplified by PCR from human spleen cDNA using forward 5′-TTAGGATCCTCTCCATCTGGTGTGCGGCTG and reverse 5′-CAAGGATCCACCTGAGCAGATGACAGCCAC primers. A cDNA fragment encoding the extracellular domain of human CD5 (residues Arg²⁵-Ser³⁴⁸; sCD5) was amplified by PCR from a template CD5-pGFP-N1 kindly provided by G. Bismuth (Institut Cochin, Paris) using forward 5′-TAGGGATCCCGGCTCAGCTGGTATGAC and reverse 5′-CTAGGATCCCGGGGTTTGGATCTTGGCAT primers. Each cDNA was inserted into the BamHI sites of the lab-modified version of pEE14 in order to obtain chimeric cDNAs encoding, in the following order, signal peptide, HA-tag, the specific protein sequence (Spα, N-SSc5D, or sCD5), a BirA recognition sequence, and 6·His tag sequences.

The sCD5, N-SSc5D, and Spα vectors were transfected into CHO-K1 cells using Lipofectamine (Invitrogen). Clones resistant to 30-μM methionine sulfoximine (MSX) were selected (21) and screened for soluble CD5 (HA-sCD5-BirA-His), N-SSc5D (HA-N-SSc5D-BirA-His), and Spα (HA-Spα-BirA-His) expression using dot blots and western blots. The best clones expressing HA-sCD5-BirA-His, HA-N-SSc5D-BirA-His, and HA-Spα-BirA-His were selected for large-scale production of protein and grown in cell factories (Nunc). Proteins secreted into tissue culture supernatants were harvested after approximately 4 weeks and purified by metal-chelate chromatography using Ni Sepharose High Performance (HisTrap HP, GE Life Sciences). HA-sCD5-BirA-His was eluted from the Ni column with 250 mM imidazole in PBS, while HA-N-SSc5D-BirA-His and HA-Spα-BirA-His were eluted with 40 mM imidazole in PBS. Fractions containing the HA-N-SSc5D-BirA-His and HA-Spα-BirA-His were further purified by anionic chromatography (UNO Q column BioRad) with 1 M NaCl. The previously produced recombinant protein sCD6 also conformed to a similar structure as the newly produced proteins, having a HA-sCD6-BirA-His sequence.

Protein purity was analyzed by SDS-PAGE (Figure S1 in Supplementary Material). Samples of the fractions obtained by chromatography were run for 1 h at 150 V, and the gels were stained with Bio-Safe Coomassie Premixed Staining Solution (Bio-Rad Laboratories) for visualization of the protein products.

For N-SSc5D immunoblotting, samples were run in SDS-PAGE for 1 h at 150 V with Tris/glycine/SDS running buffer (Bio-Rad Laboratories). Samples were transferred to the nitrocellulose membrane using the iBlot™ Gel Transfer Device (Invitrogen) following the manufacturer’s instructions. Then, the membrane was blocked with TBS, 0.1% Tween 20 (TBS-T), containing 5% non-fat dried milk, for 1 h with shaking. N-SSc5D was subsequently detected with rabbit anti-SSc5D (Abgent, 1:5,000) primary antibody in TBS-T with 3% non-fat dried milk, for 1 h at RT, followed by peroxidase-conjugated goat anti-rabbit IgG antibody (Sigma, 1:30,000) for 1 h at RT. The immunoblot was developed using ECL detection reagent (GE Healthcare Life Sciences), and the image was acquired in a ChemiDoc XRS+ system (Bio-Rad Laboratories).

Listeria monocytogenes EGD-e was grown in brain heart infusion (BHI) medium (BD-Difco) at 37°C to an optical density of 0.6 at 600 nm (OD₆₀₀; exponential phase), and Escherichia coli strains [BL21(DE3), IHE3034, RS218] were grown in LB medium at 37°C to an OD₆₀₀ of 0.45.

Recombinant proteins Spα, N-SSc5D, sCD6, and sCD5 (5 μg per assay) were incubated for 1 h at 4°C with the indicated cell suspensions of live bacteria (1 × 10⁸ cells) in binding buffer (TBS, 1% BSA, 5 mM CaCl₂). Suspensions were centrifuged at 4,000 × g for 5 min at 4°C. Cell pellets were washed thoroughly, then resuspended in 40-μl Laemmli’s sample buffer, and denatured by heating at 95°C for 10 min. Next, 20 μl of this lysate and pure recombinant proteins (25 or 100 ng) were separated in 6% SDS-PAGE. The gel was run for 1 h at 150 V with Tris/glycine/SDS running buffer (Bio-Rad Laboratories). After the SDS-PAGE, proteins were transferred to the nitrocellulose membrane using the iBlot™ Gel Transfer Device (Invitrogen) following the manufacturer’s instructions. Then, the membrane was blocked with TBS-T containing 5% non-fat dried milk, for 1 h. Cell-bound protein was subsequently detected using mouse IgG1 anti-HA (clone 16B12) from Covance (0.1 μg/ml) in TBS-T with 3% non-fat dried milk, for 1 h at RT, followed by goat anti-mouse HRP-conjugated (Santa Cruz Biotechnology) (0.02 μg/ml) in the same conditions. The immunoblot was developed using ECL detection reagent (GE Healthcare Life Sciences), and the image was acquired in a ChemiDoc XRS+ system (Bio-Rad Laboratories).

We used a laboratory four-channel SPR platform based on the wavelength spectroscopy of surface plasmons (Plasmon IV) (22) developed at the Institute of Photonics and Electronics, Czech Republic. In this SPR biosensor, the sensor response is expressed as a shift in the wavelength of SPR resonance and is directly proportional to the mass of biomolecules attached to the surface of the sensor. Using the calibration procedure described in Ref. (23), the surface density of both the immobilized receptors and the subsequently attached molecules can be determined. For an SPR resonance of around 750 nm, the shift of 1 nm in the SPR wavelength represents a change in the protein surface coverage of 17 ng/cm² (23). All the experiments were performed at 25°C. Buffers used were SA₁₀ (10 mM sodium acetate, pH 4.0/5.0), PBS (10 mM phosphate, 2.9 mM KCl, 137 mM NaCl, pH 7.4), PBNa (10 mM phosphate, 2.9 mM KCl, 750 mM NaCl, pH 7.4), and Tris (10 mM Tris-HCl, pH 7.4).

The sensor chip was functionalized with a mixed self-assembled monolayer (SAM) by incubating the cleaned gold chip in degassed absolute ethanol with a mixture (7:3) of HSC₁₁(EG)₄OH and HSC₁₁(EG)₆OCH₂COOH alkanethiols at a final concentration of 200 μM. The HSC₁₁(EG)₆OCH₂COOH alkanethiols terminated with a carboxylic head group were used to anchor a receptor by amino coupling, while HSC₁₁(EG)₄OH alkanethiols terminated with hydroxylic group were used to form a stable non-fouling background. For that purpose, the sensor chip was immersed in a mixed thiol solution at a temperature of 40°C for 10 min and then stored overnight in the dark at RT. After the formation of the mixed SAM, the chip was removed from the solution, rinsed with absolute ethanol and deionized water, and dried with nitrogen. The chip was then immediately mounted to the prism on the SPR sensor. The activation of carboxylic terminal groups was performed in situ by injecting deionized water followed by a 1:1 mixture of NHS and EDC for 5 min and deionized water again.

Conditions for immobilization have been optimized in terms of running buffer composition and pH, as well as sufficient surface coverage. Immobilization of proteins via covalent attachment to COOH/OH SAM was performed at a flow rate of 30 μl/min and a temperature of 25°C. To immobilize the receptors, sodium acetate (SA₁₀) pH 4.0 (Spα, N-SSc5D, and sCD6) or 5.0 (sCD5) was flowed trough the activated surface until a baseline was achieved. Then, the SA₁₀ solutions containing the receptors (2–5 μg/ml) were flowed across the activated surface until a desired surface coverage was achieved. To remove the non-covalently bound receptors, the high ionic strength PBNa buffer was flowed along the sensor surface. Finally, the sensor surface was treated with 1 M EA to deactivate residual carboxylic groups.

Bacteria cells were pelleted by centrifugation (4,000 × g, 5 min) and resuspended in PBS. Then, to preserve bacterial cell morphology and to increase the sensitivity of the detection, cell aliquots were exposed to isopropanol (final concentration, 70% v/v) for 20 min at RT. The pellets of isopropanol-fixed cells were obtained by centrifugation at 7,000 × g for 5 min and washed twice with PBS. Next, running buffer was flowed along the sensor surface until the baseline was achieved. Bacteria were resuspended in the running buffer at a concentration of 1 × 10⁷ CFU/ml (or as indicated in the text) and delivered at a flow rate of 50 μl/min to the sensor surfaces with the immobilized proteins. Then, the running buffer was introduced again. The binding of bacteria to the sensor surface was detected as the difference in the sensor response between the equilibrium level after washing the bound surface with the running buffer and the baseline level obtained before the injection of the bacteria solution.

In this work, we used reference-compensated measurements and tested several different surfaces to be used as a reference surface. These included a surface without receptors, surfaces covered with blocking molecules such as BSA, casein, or NeutrAvidin, and a surface with immobilized reference protein (sCD5). The study revealed that there was considerable adsorption of bacteria to a bare alkylthiolate SAM (used as a functional layer) without any receptors/molecules immobilized and that the binding of bacteria to the surface coated with blocking molecules was significantly higher than that observed in case of surface coated with a reference protein. Therefore, this approach was selected as the best option.

We first assessed the binding of the SRCR-containing extracellular domains of Spα, SSc5D, CD6, and CD5 (respectively, Spα, N-SSc5D, sCD6, and sCD5) to E. coli strains BL21(DE3), IHE3034, and RS218, and to L. monocytogenes strain EGD-e, using conventional bacteria-protein binding assays. Although Spα, sCD5, and sCD6 had previously been tested for bacteria binding (6, 9, 10), no experiments had been performed for SSc5D. We incubated 5 μg of each recombinant SRCR protein with bacterial suspensions of 1 × 10⁸ live cells (colony-forming units, CFU) at 4°C, followed by centrifugation and immunoblotting of the pelleted bacteria.

We confirmed the interaction of recombinant Spα with all bacterial samples tested, having an enhanced capacity to bind E. coli RS218 comparing with the other bacteria strains (Figure 1). However, and in contrast with previous studies, no detectable sCD6 was recovered in association with the bacterial pellets, using our experimental setup. There was also no bacteria-bound sCD5 detected, but this was expected, given that CD5 was reported not to bind to bacteria (10). As observed from the experiments, N-SSc5D distinctly detected E. coli RS218 and IHE3034, although there was no conclusive evidence at this stage that it could bind to E. coli BL21(DE3) or to L. monocytogenes.

The results from the previous experiment suggested that different SRCR proteins had distinct binding properties to different bacterial strains, which might not have been highlighted in previous publications, each addressing a different SRCR protein at a time. Aware that western blot detection might not be the most sensitive method to emphasize these differences, we designed a new SPR-based assay to enhance the sensitivity of detection of extracellular proteins binding to bacteria. In this assay, the proteins are directly attached to the sensor chip by amine coupling. Suspensions of isopropanol-fixed bacteria, resuspended at a concentration of 1 × 10⁷ CFU/ml or lower, are then delivered to the sensor surface containing the immobilized proteins. The output of the SPR sensor (expressed in nanometer of resonant wavelength) is directly proportional to the amount of biomolecules attached to the active surface of the sensor. The difference in the sensor output before injection of bacteria (baseline level) and after washing the surface with captured bacteria in buffer is therefore proportional to the amount of bacteria captured (irreversibly bound) by the proteins immobilized on the sensor surface. This quantity was used to characterize the ability of the respective immobilized proteins to bind bacteria.

We considered the following as references for the binding spectra: (a) the positive interaction of Spα with neuropathogenic E. coli K1 RS218 and with L. monocytogenes EGD-e and (b) the null interaction of sCD5 with both bacteria species (Figure 2A). Spα and sCD5 were immobilized in alternate flow chambers, bacteria were injected at 1 × 10⁷ CFU/ml, and SPR plots registered.

Next, we tested whether the interactions of N-SSc5D with E. coli RS218 and L. monocytogenes EGD-e were measurable by SPR. As illustrated in Figure 2B, the interaction levels of N-SSc5D with bacteria were lower than those of Spα in both cases (between 15 and 40% across several experiments), but quite distinct from the profiles obtained for sCD5. These results confirmed the WB detection of the N-SSc5D-E. coli RS218 interactions seen in Figure 1, but further advanced in the detection of a subtle interaction between N-SSc5D with L. monocytogenes.

The results were reliable and qualitatively consistent among experiments, with only small variations in the absolute values of the responses. The chip-to-chip reproducibility of the interaction was >82% and >95% for N-SSc5D and sCD5 binding, respectively. The reproducibility values were determined from three independent experiments for each protein.

To test whether N-SSc5D could have a different capacity to bind different E. coli strains, we immobilized N-SSc5D and simultaneously injected, in separate flow channels, the non-pathogenic laboratory BL21(DE3) strain, and the meningitis-causing RS218 and IHE3034 E. coli strains. As another control of null-binding, we used in the fourth flow channel, heat-killed IHE3034. In parallel, we performed the same experiment with immobilized Spα. As seen in Figure 3, E. coli RS218 gave the best binding curve to N-SSc5D, followed by IHE3034, and finally BL21(DE3). Heat-killed IHE3034 only marginally bound to N-SSc5D, suggesting that the bacterial determinants recognized by N-SSc5D are destroyed by heat. The binding profile of Spα to the different E. coli strains was not too different, binding marginally better to RS218 and BL21(DE3) than N-SSc5D, and less to IHE3034 than N-SSc5D, indicating that these proteins have slightly distinct recognition profiles but can nevertheless distinguish between different bacterial strains.

To directly assess the differential binding capacity of the different SRCR receptors to a same bacterial preparation, we immobilized Spα, N-SSc5D, sCD6, and sCD5 in the four sensing channels and simultaneously injected E. coli RS218 at 1 × 10⁷ CFU/ml to all channels. As depicted in Figure 4A, RS218 bound with the highest level to Spα, followed by N-SSc5D. As expected, sCD5 displayed the lowest level of RS218 binding; however, binding of the bacteria to immobilized sCD6 was, although relatively low, noticeably higher than that binding to sCD5. This indicates that despite the apparent negative result of Figure 1, there is some above-background level of binding of sCD6 to E. coli RS218 measurable by this SPR-based method.

Finally, we evaluated the sensitivity of the method by analyzing the interaction of E. coli RS218 with N-SSc5D using suspensions with decreasing bacteria concentration. Figure 4B represents again the profiles of binding of E. coli RS218 at 1 × 10⁷ CFU/ml to immobilized N-SSc5D and sCD5. Then, the specific binding was obtained by subtracting the signals arising from the measuring channels with immobilized N-SSc5D from those measured in the sCD5-immobilized reference channels. Three different concentrations of bacteria were used, 3, 5, and 10 × 10⁶ CFU/ml, and for each concentration, the subtractive plots are represented in Figure 4C, indicating that the method clearly detects specific binding of E. coli RS218 to N-SSc5D even when using bacteria concentrations as low as 3 × 10⁶ CFU/ml.