Identification of the immunodominant regions of Staphylococcus aureus fibronectin-binding protein A

Abstract

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional 'microbial surface components recognizing adhesive matrix molecules' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F130-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F130-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F130-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.

Figure 1. Immunization schedule and expression of recombinant truncated fragment proteins.

(A) Diagrams showing the experimental design of the immunization schedule for the measurement of antibodies, the survival rates up to 14 days after bacterial challenge and the bacterial budens on days 1 and 3 after bacterial infection. (B) Structural organization of the fibronectin-binding protein, FnBPA from S. aureus strain MRSA252 and schematic diagram illustrating the primary structure of the FnBPA_1-965, F1_30-500(F1) and F2_501-941(F2). (C) Recombinant GST-tagged F1 and F2 were purified by affinity chromatography and analyzed by SDS-PAGE.

Figure 2. Production of anti-F1 and F2 antibody by BALB/c mice immunized with F1 or F2.

The anti-serum was collected at the first week after the last immunization. Each group has six mice. (A) Elisa titration of antibodies directed against F1 in sera from mice immunised with F1 and alum or PBS and alum. The ELISA plates were coated with F1 as antigen. (B) Elisa titration of antibodies directed against F2 in sera from mice immunised with F2 and alum or PBS and alum. The ELISA plates were coated with F2 as antigen. Standard deviations are indicated by bars.

Figure 3. Immunization with the recombinant protein vaccine (F1) generated protective immunity against MRSA252 challenge.

BALB/c mice (n = 15) were immunized with individual antigens (F1, F2) and alum adjuvant. The animals were challenged by intravenous injection of MRSA252 (1×10⁹ CFU) and were monitored for 14 days. Compared with animals receiving antigen-free PBS and the adjuvant alone, the significance of the protective immunity generated by the various antigens was measured with a log rank test: F1, P = 0.0038; F2, P = 0.5375. The asterisks represent a statistically significant difference (**P<0.01). Representative results from one of three independent experiments are shown.

Figure 4. Generation of the fragments of F1 and F2.

(A to B) A total of 12 different fragments were produced as GST fusion proteins from the fulllength FnBPA. (C to D) The quality of the GST fusion proteins, F1_30-500, F1A_30-173, F1B_110-263, F1C_195-333, F1D_264-372, F1E_373-500, F2_501-941, F2A_501-616, F2B_586-756, F2C_663-865, F2D_738-900, and F2E_805-941, was monitored in an SDS-polyacrylamide gel stained with coomassie blue dye. The molecular masses are shown on the left.

Figure 5. Reactivity of FnBPA fragments with mouse antisera.

An ELISA plate was coated with the FnBPA fragments in the form of GST fusion proteins (displayed along the x axis) and reacted with each of the 20 mouse antisera at a dilution of 1∶1,000 (y axis). (A) The OD values obtained from the reactions of each fusion protein with the 20 mouse (immunization with F1 protein) antisera are expressed as means and standard deviations (y axis). (B) The OD values obtained from the reactions of each fusion protein with the 20 mouse (immunization with F2 protein) antisera are expressed as means and standard deviations (y axis).

Figure 6. Immunization with the recombinant protein vaccine F1B_110-263(F1B) generates protective immunity against systemic MRSA infection.

BALB/c mice (n = 15) were immunized with individual antigens (F1, F1B) and alum adjuvant. The animals were challenged by intravenous injection of two different MRSA strains and were monitored for 14 days. Compared with animals receiving antigen-free PBS and the adjuvant alone, the significance of the protective immunity generated by the various antigens was measured with a log rank test. (A) S. aureus strain MRSA252 (challenge dose, 1×10⁹ CFU); (B) S. aureus strain WHO-2 (challenge dose, 1×10⁹ CFU). The asterisks represent a statistically significant difference (*P<0.05, **P<0.01, ***P<0.001). Representative results from one of three independent experiments are shown. (C)Bacterial numbers in kidneys of immunized and control mice were determined at 1and 3 days after infection with 2.5×10⁸ CFU i.v. Each group included 5 mice. Data are presented as box plots, and the medians and interquartile ranges are shown. Asterisks indicate significant differences between vaccinated and control mice (** P<0.01).

Figure 7. Opsonic activity of antibodies to FnBPA against the S. aureus MRSA252.

S. aureus MRSA252 (1–2×10⁵ CFU per ml) was incubated in the presence of live leukocytes (1–2×10⁶ HL-60 cells per ml) and diluted rabbit antisera against F1, F1B_110-263 or normal rabbit sera (NRS) in the presence of infant rabbit complement. They were plated on agar medium to measure bacterial survival as determined by CFU after 90 minute incubation. Then the percent of killing was calculated. The data shown are the means and the standard error of the means derived of 3 to 5 independent experiments. Unpaired 2 tailed student's t- tests were perfomed to analyze the statistical significance of data comparing non-reactive rabbit anti-serum with rabbit serum raised against specific antigens F1 (***P<0.001) and F1B_110-263 (***P<0.001).