Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain

Abstract

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Fig. 1

Schematic representation of the Streptococcus mutans P1 protein (a) and the recombinant gene encoding the truncated P1_39–512 polypeptide (b) cloned in Bacillus subtilis pLDV701 vector. The predicted molecular mass of the recombinant P1_39–512 protein is indicated on the right of (b).

Fig. 2

Detection and purification of the recombinant P1_39–512 protein expressed by Bacillus subtilis cells. (a) Immunodetection of P1 or P1_39–512 expressed by Streptococcus mutans PC3370C (1P1) or B. subtilis LDV701 (LDV701) strains, respectively. Negative controls include S. mutans PC3370 (ΔP1) and the B. subtilis LDV700 strain (LDV700). − and + denote cells induced or not with IPTG, respectively. Estimated molecular weights (kDa) of the reactive protein bands are indicated. (b) Immunodetection of recombinant P1_39–512 in the soluble (S) or insoluble (I) fractions of the B. subtilis LDV701 following induction with IPTG. (c) Purification of recombinant P1_39–512 expressed in B. subtilis. Imidazole concentrations used to elute the protein from the nickel-containing resin are indicated. Proteins were separated in 12.5% polyacrylamide gels and stained with Comassie Brilliant Blue. MW, molecular weight markers indicated in kilodaltons (kDa) at the right side of the figure.

Fig. 3

Antigen specificity of antibodies raised in mice immunized with P1_39–512. (a) Western blots developed with serum samples collected from mice immunized with P1_39–512 or (b) P1_39–512d. Samples: 1,Streptococcus mutans PC3370C strain; 2, S. mutans PC3370 strain; 3, Bacillus subtilis LDV700 strain following incubation without IPTG; 4, B. subtilis LDV700 strain following incubation with IPTG; 5, B. subtilis LDV701 strain following incubation without IPTG; 6, B. subtilis LDV701 strain following incubation with IPTG; 7, recombinant P1 protein; 8, P1_39–512 purified protein. Proteins were sorted in 12.5% polyacrylamide gels. The molecular weights of the reactive protein bands are indicated. (*) and (**) indicate the molecular weights of P1 and P1_39–512 proteins, respectively.

Fig. 4

IgG titration in antisera raised in mice immunized with full-length P1, expressed in Escherichia coli, and with P1_39–512 or P1_39–512d, produced in recombinant Bacillus subtilis. Serum dilutions were reacted in ELISA plates coated with P1_39–512 (closed symbols) or heat-denatured P1_39–512d (open symbols). Symbols: (circles) anti-P1; (squares) anti-P1_39–512; and (triangles) anti-P1_39–512d.

Fig. 5

Immunofluorescence labeling of Streptococcus mutans cells using antisera from mice immunized with P1_39–512 or P1_39–512d. Samples: (a) S. mutans PC3370C strain reacted with the anti-P1_39–512 antiserum; (b) S. mutans PC3370C strain reacted with anti-P1_39–512 d antiserum; (c) S. mutans PC3370 (ΔP1) strain reacted with anti-P1_39–512 antiserum; (d) S. mutans PC3370 (ΔP1) strain reacted with anti-P1_39–512d serum. Labeled S. mutans cells were revealed with fluorescein isothiocyanate -conjugated goat anti-mouse IgG.

Fig. 6

Functional characterization of antibodies raised in mice immunized with P1_39–512 produced in Bacillus subtilis. (a) Inhibition of saliva-mediated aggregation of Streptococcus mutans PC3370C cells. Samples containing bacterial cells and the tested serum were incubated for 5 min at 37 °C and the OD_600nm was measured (T0). After an additional 60-min incubation period at 37 1C, the OD (T60) was measured once more. The values are expressed as the difference between T0 and T60 for each tested serum dilution. Symbols: (◆) nonimmune serum; (●) anti-P1 serum; (■) anti-P1_39–512 serum; (▲) anti-P1_39–512d serum; (□) anti-P1 serum adsorbed with P1_39–512; and (△) anti-P1 serum adsorbed with P1_39–512d. (b) Inhibition of S. mutans adherence to immobilized saliva in microplate wells. Streptococcus mutans PC3370C cells were mixed with diluted tested serum samples, incubated for 30 min to 37 °C, and transferred to microplate wells coated with clarified saliva and incubated for an additional 2 h. Bound cells were revealed by staining with 0.5% crystal violet and determination of absorbance of the solubilized cell-bound stain at 600 nm. Symbols are the same as those shown in (a) and (◇) cells incubated in the presence of 3 mM EDTA. All tested sera were diluted in order to reach a final titer of 10⁴. Data represent the average of three independent experiments. Values are expressed as mean ± SD.