Escherichia coli BL21 (Thermo Fisher, Shanghai, China) was used as competent cell for recombinant plasmid transformation and expression of the target protein. pET28a-inaQn-P (GII.4) and pET28a-P (GII.4) were constructed as previously described (Niu et al., 2015), pET28a-inaQn was constructed by inserting inaQn into Nco I/BamH I digested pET28a and used as a control. All the recombinant plasmids were used to transform bacteria E. coli BL21. Figures 1A,B presented for the plasmid DNA map and a schematic figure for displayed P protein on the surface of pET28a-inaQn-P(GII.4) transformed bacteria [INP-P (GII.4) BL21].

Recombinant E. coli BL21 harboring inaQn and P protein fusion gene [named INP-P (GII.4) BL21], P protein gene [named P (GII.4) BL21] and inaQn gene (named INP BL21) were cultured as previously reported (Niu et al., 2015). Cells were cultured in 5.0 ml of LB medium containing 100 μg/ml kanamycin with shaking (220 rpm) at 37°C, overnight. The cells (50.0 μl) were subcultured in 5.0 ml fresh LB medium with 60 μg/ml kanamycin. When the OD₆₀₀ reached 0.5, isopropyl-β-D-thiogalactopyrano-side (IPTG) was added to reach a final concentration of 0.4 mmol/l. The cells were incubated at 25°C for 12 h, then washed and diluted to OD₆₀₀ = 1.0 with sterile PBS, and then kept at 4°C for further use.

The localization of surface-displayed P protein (SD-GII.4P) was examined using immunofluorescence microscopy (Olympus, Japan) as previously described (Li et al., 2009). Anti-VP1 (GII.4) polyclonal antisera from immunized Balb/C mice (1:5000 in PBS) were used as the primary antibody (Niu et al., 2015), and 1:100-diluted FITC-conjugated goat anti-mouse IgG (Thermo Fisher, USA) was used as the secondary antibody. The anti-VP1 (GII.4) polyclonal antisera were pre-absorbed with E. coli BL21 extract to reduce potential non-specific. Three sets of negative controls were used. P (GII.4) BL21, INP BL21, and E. coli BL21 were used to test non-specific binding of polyclonal antisera. INP-P (GII.4) BL21 without primary antibody (with PBS only) was used as additional negative control for non-specific binding of FITC-conjugated secondary antibody.

Romaine lettuce was collected randomly from a local grocery store and stored at 4°C. The leaves were washed three times with sterile distilled water. For microscopy, approximately 2 cm² of the strips were cut from the ends of leaves using a sterile blade. For ELISA, the romaine lettuce was made into leaf extract (LE) and vein extract (VE), collectively referred to as RE. LE was prepared from the top half of a romaine leaf (15 g), while VE was prepared from the excised main veins. The same volume of PBS (wt/vol) was added to each vegetable matter sample, and blended for 5 min at 4°C. The liquid phase was transferred to a sterile microcentrifuge tube and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was transferred to a fresh sterile tube and kept at 4°C for further use.

Human saliva was collected from A, B, O blood type volunteers and treated according to the previous report, with minor modification (Wang et al., 2014). The secretor status of individual saliva was indirectly determined by an ELISA assay for its ability to bind purified bacteria-expressed GI and GII VP1 and P domains (Niu et al., 2015) and directly determined with corresponding monoclonal antibodies. Although HuNoV binding ability was confirmed in all saliva tested, three saliva samples from the same blood type were mixed and used for competitively assays. The study was approved by the Institutional Bio-safety Committees (IBC) of College of Agriculture and Biology, Shanghai Jiao Tong University, and written informed consent was obtained from the volunteers. Briefly, each type of saliva was collected from at least three volunteers and mixed. Then, each saliva sample was boiled for 5 min, and then followed with centrifugation at 10,000 × g for 5 min at 4°C. The individual supernatant of saliva from each blood group was mixed, aliquoted and stored at –20°C for further use. Saliva from non-secretors was used as negative control.

Strips of romaine lettuce on glass slides were blocked with 1% bovine serum albumin (BSA, Yeasen, Shanghai, China) in PBS at 4°C, overnight, and then soaked in sterile PBS for 5 min. The strips were moved into a sterile plate containing INP-P (GII.4) BL21 or E. coli BL21 (negative control), and incubated at room temperature for 1 h. The strips were then washed with PBS solution and put on a slide. Then, 70 μl of primary antibody (Niu et al., 2015) at a dilution of 1:2,000 in PBS was added onto the surface of strips and incubated at room temperature for 1 h. After incubation, strips were washed three times with PBS. A volume of 50 μl FITC-conjugated goat anti-mouse IgG (H+L) (Yeasen, Shanghai, China) at a dilution of 1:100 in PBS was added onto the strips, and incubated at room temperature for 1 h. After washing 3 times with PBS, 30 μl PBS was applied to the surface of the romaine, covered with a coverslip, and observed under a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). The images were magnified at 700×.

The RE-binding enzyme-linked immunosorbent assay (ELISA) was performed as previously reported (Gandhi et al., 2010), with minor modification. Briefly, LE or VE was diluted in PBS (1:5, pH = 7.4) (Gandhi et al., 2010), and PGM (Sigma, MI, USA) was dissolved in carbonate buffer solution (pH = 9.6) to 1.0 mg/ml (Wang et al., 2014). Then, LE, VE, or PGM was added into and allowed to coat 96-well microtiter plates (100 μl per well) at 4°C overnight. Plates were washed three times with PBS, and blocked with 1% BSA at 37°C for 2 h. Plates were washed three times with PBS containing 0.1% Tween-20 (PBS-T). The INP-P (GII.4) BL21, E. coli BL21, P (GII.4) BL21, and INP BL21 samples (100 μl) were added to each well, respectively, and incubated at 37°C for 1 h. After washing three times with PBS-T, 100 μl of primary antibodies (1:10,000 in PBS) was added to each well, and incubated at 37°C for 1 h. After washing three times with PBS-T, 100 μl of peroxidase-conjugated goat anti-mouse IgG (H+L) (Yeasen, Shanghai, China) at a dilution of 1:5,000 in PBS was added to each well, and incubated at 37°C for 1 h. After washing five times with PBS-T, 100 μl 3,3′,5,5′-tetramethylbenzidine (Yeasen, Shanghai, China) was added to each well. Plates were kept in the dark for 5 min, and then 50 μl of 2.0 mol/l H₂SO₄ was added to stop the reaction. OD₄₅₀ value was measured using a Microplate Reader (Sanjose, Shanghai, China). In addition, to determine if INP could bind to RE, INP BL21 was added to RE coated wells, followed by antibodies against INP (prepared as described in Li et al., 2009) and peroxidase-conjugated secondary antibody.

Romaine extract-coated or PGM-coated wells with PBS were used as the blank control. The bacteria E. coli BL21 was used as a negative control (N). Samples were considered as positive when the positive to genitive (P/N) ratio was greater than 2.0.

Boiled mixed saliva was diluted five times with PBS. The INP-P (GII.4) BL21 (100 μl) cells were incubated with the diluted boiled saliva (100 μl) or 1.0 mg/ml PGM (100 μl) solution at 37°C for 30 min. After centrifugation at 10,000 × g for 2 min at 4°C, the precipitated INP-P (GII.4) BL21 was re-suspended in PBS (pH = 7.4). The INP-P (GII.4) BL21 incubated with PBS was used as untreated control. E. coli BL21 (negative control) was treated with the same process. After blocking, the INP-P (GII.4) BL21 was detected by RE-binding based ELISA as described in the Section “Romaine Lettuce Extract-Binding or PGM-Binding Based ELISA”. To calculate percentage inhibition, the P/N ratio of untreated samples was equal to 0% inhibition and P/N ratio of 2.0 was equal to 100% inhibition.

Porcine gastric mucin was used as a positive control for protein denaturation and carbohydrate oxidation (Tian et al., 2005, 2007). LE or VE was boiled for 5 min, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was diluted five times with carbonate buffer solution (pH = 9.6), and was used to coat the wells as described in Section “Romaine Lettuce Extract-Binding or PGM-Binding Based ELISA”. To determine whether carbohydrates were involved in INP-P (GII.4) BL21 binding, 100 μl 4.0 mg/ml NaIO₄ (Adamas Reagent Co., Ltd., Shanghai, China) was added to wells coated with LE or VE (Esseili et al., 2012). The plates were incubated at 37°C for 30 min prior to the addition of the INP-P (GII.4) BL21. PGM, LE, or VE without protein denaturation or oxidation was used as untreated controls. After treatment, the binding capability and percentage of inhibition were evaluated as described in Sections “Romaine Lettuce Extract-Binding or PGM-Binding Based ELISA” and “Competitive Inhibition ELISA Assay”.

The INP-P (GII.4) BL21 and E. coli BL21 (negative control) was diluted across a range of PBS of different pHs, and adjusted to the same OD₆₀₀ value. The INP-P (GII.4) BL21 was detected by RE-binding-based ELISA as described in Section “Romaine Lettuce Extract-Binding or PGM-Binding Based ELISA”. P/N ratio at pH 6.4 was equal to 100% to calculate the percentage of binding.

Each experiment (N) was performed in triplicate (n = 3) and repeated at least three times (N > 3). The means and standard deviations from independent experiments were presented in all figures. One-way analysis of variance (ANOVA) was utilized for data comparison. Differences in means were considered significant when the p < 0.05.

To verify the surface localization of SD-GII.4P on INP-P (GII.4) BL21 cells, immunofluorescence assay was performed (Figure 2). SD-GII.4P fusion proteins were clearly visualized as fluorescent spots on the transformed cell surface (Figures 2A,B). No FITC fluorescence signals were observed in P (GII.4) BL21 (Figures 2C,D), INP BL21 samples (Figures 2E,F) and E. coli BL21 (data not shown).

The INP-P (GII.4) BL21 bacteria were observed directly by Confocal Laser Scanning Microscope using FITC-labeled antibodies. After being washed, the INP-P (GII.4) BL21 bacteria were found to be distributed on the leaf (Figure 3A), stomata (Figure 3B) and vein (Figure 3C) of romaine lettuce. Romaine lettuce with E. coli BL21 bacteria exhibited no green fluorescent signals on its surface (Figures 3D–F). The red signals were autofluorescence of chloroplasts (Figure 3).

The OD₄₅₀ reading of E. coli BL21 was used as control N to calculate P/N ratio in RE binding based ELISA and inhibition assays. The P/N ratio of P (GII.4) BL21 and INP BL21 were all around 1.0 for LE and VE with antibodies against VP1 of GII.4 HuNoV (Figure 4). There were no significant differences between these two samples and E. coli BL21. The result suggested that BL21 and InaQN could not bind to RE measured by HuNoV specific antibodies. In addition, with InaQN specific antibodies, we demonstrated that INP BL21 could not bind to RE coated wells as its P/N ratio was closed to E. coli BL21 control (data not shown). We further demonstrated that HuNoV P proteins expressed in P (GII.4) BL21 were not be able to bind RE due to its nature of intracellular expression. However, INP-P (GII.4) BL21 could bind to wells coated with LE or VE. The P/N ratios ranged from 2.38 to 4.01 for LE and 2.40 to 4.21 for VE. A slight stronger but not significant binding of INP-P (GII.4) BL21 was observed in VE (3.21 ± 0.92) than that of LE (3.09 ± 0.84). The average inhibition rates were 92.68 ± 11.27%, 95.64 ± 4.46%, 62.98 ± 12.25%, and 8.31 ± 6.57% when INP-P (GII.4) BL21 was pre-incubated with type A, O, B saliva, or PGM (Figure 5) prior to being added to LE-coated wells. Similarly, the average inhibition rates were 91.32 ± 12.31%, 89.55 ± 13.37%, 70.80 ± 15.37%, and 23.36 ± 8.11% when INP-P (GII.4) BL21 was pre-incubated with type A, O, B saliva, or PGM prior to being added to VE-coated wells (Figure 5). The binding of INP-P (GII.4) BL21 to both LE and VE could be completely inhibited by type-A and -O saliva, and partially inhibited by PGM and type-B saliva. There was no significant difference between the binding of INP-P (GII.4) BL21 to leaf and vein areas of romaine lettuce. In addition, the binding between INP-P (GII.4) BL21 and LE or VE could not be inhibited by saliva from non-secretors (data not shown).

To determine the nature of the receptor/ligand of romaine lettuce involved in binding INP-P (GII.4) BL21, LE, and VE were either boiled to denature proteins, or oxidized by NaIO₄ to remove the functional groups of carbohydrates. It has been reported that HBGAs in PGM were involved in the binding of HuNoV (Tian et al., 2005). Therefore, PGM was used as positive control for the assay. The effects of protein denaturation and carbohydrate oxidation on LE and VE were similar to that of the same to PGM. Wells coated with NaIO₄-oxidized LE, VE or PGM resulted in complete inhibition of binding with INP-P (GII.4) BL21 with inhibition rates of 95.49 ± 7.81%, 100%, and 100%, respectively (Figure 6). A significant difference in binding inhibition was observed between NaIO₄-treated and untreated reactions with the three groups, but no significant difference was observed between the three groups (p > 0.05). Partial inhibition was observed when INP-P (GII.4) BL21 was applied to wells coated with LE, VE or PGM treated with heat to denature proteins (Figure 6). When PGM, LE and VE were heat-treated the inhibition rates were 21.48 ± 11.09%, 32.23 ± 1.76%, and 44.66 ± 12.36%, respectively. There was a significant difference between these three groups treated with or without heat-treatment but no significant difference among these three groups (p > 0.05). These results demonstrated that carbohydrates rather than proteins played an important role in LE and VE for HuNoV binding.

To determine the effect of pH on the binding of INP-P (GII.4) BL21 bacteria to romaine lettuce, especially in the vicinity of isoelectric point (pI) of the P domain protein (pI = 6.4), LE or VE were treated at conditions below, above, and at pI of P protein (Figure 7). PGM was used as a control. Aside from a significant reduction of binding between INP-P (GII.4) BL21 to VE at pH 5.4 (p < 0.004), there was no significant difference between binding of INP-P (GII.4) BL21 bacteria to PGM, LE, or VE at various pHs tested.