Dissimilar properties of two recombinant Lactobacillus acidophilus strains displaying Salmonella FliC with different anchoring motifs

Abstract

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

Fig. 1.

Gene map of pTRK1033 and pTRK1034.

Fig. 2.

Expression and cell surface display of FliC by recombinant L. acidophilus strains. (a) Concentrated culture supernatants (Sup) and cell extracts (Cell) were analyzed by Western blotting using an anti-FliC monoclonal antibody. Flagellin prepared from Salmonella serovar Typhimurium was also applied as a reference protein. Molecular sizes are shown on the left. (b) Cell surface proteins extracted with either PBS or 8 M urea were subjected to Western blotting for the detection of FliC. (c) FliC located on the cell surface was detected by flow cytometry. The fluorescence of PE represents surface-associated FliC. MFI, mean fluorescence intensity.

Fig. 3.

Relative amounts of surface-associated FliC. Recombinant L. acidophilus cell suspensions (dilution 0, 1 × 10⁹ CFU/ml) were serially diluted (dilution 1, 5 × 10⁸ CFU; dilution 2, 2.5 × 10⁸ CFU; and so forth) and were incubated with anti-FliC monoclonal IgG (dilution, 1:50,000). The remaining anti-FliC antibody that was not absorbed by the bacteria was detected by ELISA. The data represent three separate experiments, and mean values for duplicate samples are shown. A₄₅₀, absorbance at 450 nm.

Fig. 4.

TLR5-stimulating activities of recombinant lactobacilli NCK1895, NCK2158, and NCK2160. The activity of SEAP released from HEK293 cells was measured after incubation with the recombinant bacteria. Values are means + standard errors from 3 experiments.

Fig. 5.

TLR5 expression by human myeloid DCs stimulated with recombinant lactobacilli. Flow cytometry was used to analyze TLR5 expression on the DCs. (a) The isolated DCs were stained before (0 h) or after (24 h) incubation with or without stimulation. (b) TLR5 expression levels were compared after 24 h of incubation. MFI, median fluorescence intensity. *, P < 0.01.

Fig. 6.

Maturation of human myeloid DCs by stimulation with FliC-displaying L. acidophilus strains. Freshly isolated myeloid DCs from 6 different donors were incubated with or without recombinant lactobacilli for 24 h. The stimulated DCs were collected and stained with fluorescently labeled anti-CD83, anti-CD86, anti-CD80, and anti-CD40 antibodies. The fluorescence intensities of the stained DCs were determined by flow cytometry. MFI, median fluorescence intensity. *, P < 0.01.

Fig. 7.

Analysis of multiple cytokines secreted by lactobacillus-stimulated DCs. Freshly isolated human myeloid DCs (2 × 10⁵ cells in 0.2 ml) were incubated with or without recombinant lactobacilli (2 × 10⁶ CFU in 0.2 ml) for 24 h. Culture supernatants were collected and analyzed with a Milliplex MAP kit. The concentration (in picograms per milliliter) of each cytokine is shown along the y axis. The values for IL-8 and IFN-α2 are expressed as MFI because these numbers were out of the range of standard curves. *, P < 0.01.

Fig. 8.

Sensitivities of recombinant bacteria and FliC to simulated digestive juice. (a and c) Recombinant L. acidophilus cells were incubated with simulated gastric juice (a) or simulated small intestinal juice (c). Viable cells (colony-forming cells) at several time points were counted. (b and d) Cell surface FliC was detected by dot blot analysis after treatment with either simulated gastric juice, including different concentrations of pepsin (b), or simulated small intestinal juice, including different concentrations of pancreatin (d).

Fig. 9.

Protective effects of bicarbonate and SBTI against simulated digestive juices. Recombinant lactobacilli were suspended in either bicarbonate buffer or SBTI solution prior to treatment with simulated digestive juice. Viable cells and surface FliC were detected after 90 min of incubation with either simulated gastric juice (a and b) or simulated small intestinal juice (c and d).