Fig 1.
Construction and purification of the recombinant FSBM protein.
(A) The map of the pET24a-fsbm plasmid and the sequence of the synthetic fsbm gene. The plasmid was transformed, and the expression of rFSBM was induced. The recombinant protein was purified as described in Materials and Methods. Purified recombinant rFSBM protein was separated by SDS-PAGE (B) and the antigenic epitope of C-terminal domain of SPE B within rFSBM protein was analyzed by Western blotting (C) and ELISA (D) using the anti-SPE B antibody.
Fig 2.
Immunization of recombinant FSBM protein protected mice against invasive emm1 strain GAS-induced death.
(A) The average titers of rFSBM-specific serum IgG collected from BALB/c mice that were immunized with recombinant FSBM proteins alone or rFSBM proteins combined with Freund's Adjuvant or AddaVax. (B) The survival rates of mice immunized with the rFSBM protein combined with different adjuvants were significantly higher than the adjuvant controls, including the mice immunized with CFA adjuvant only or immunized with AddaVax only controls, post subcutaneously challenged with 2 x 108 CFU of emm1 strain GAS. The survival curves were compared for significance using the log-rank test for the immunized mice compared to the adjuvant-only control groups (P < 0.01).
Fig 3.
Specific reactivity of the rFSBM–immunized serum with their relative antigens.
The rFSBM-immunized serum recognized purified SPE B (A), commercial streptolysin S (B), synthetic peptides of Sfb1 (C), and the pepM proteins collected from different emm types of GAS (D). *P < 0.05, **P < 0.01, compared with values determined for the CFA adjuvant only control serum. The SPE B or the pepM proteins purified from the culture medium of wild-type GAS, speB-mutant, or the emm6 deletion mutant were determined by Western blotting (E) as described in Materials and Methods.
Fig 4.
In vitro opsonization and macrophage-associated bacteria were enhanced by the rFSBM antiserum.
(A) The M protein-specific reactivity of rFSBM-immunized sera was determined by flow cytometry with emm6 wild type GAS and the emm6-deletion mutant, as described in Materials and Methods. (B) Sera from rFSBM-immunized mice or CFA adjuvant-only control mice were incubated with the wild type emm6-type GAS or the emm6-deletion mutant for 1 h, and then serum-treated GAS was taken to infect RAW 264.7 cells at a MOI of 25 for 1 h. The unbound bacteria were removed by washing with sterile PBS and the total number of bacteria bound with the cells and intracellular bacteria were quantified by plating on THY agar plates as described in Materials and Methods. The macrophage-associated bacterial number of anti-rFSBM serum-treated wild type GAS was increased compared to that of the CFA adjuvant-only control serum-treated GAS. The results of three experiments are shown and expressed as the mean ± SD. *P< 0.05, compared with the values determined for the control serum-treated emm6 wild type GAS.**P< 0.01, compared with the values determined for the PBS-treated emm6 wild type GAS or the anti-rFSBM serum-treated emm6-deletion mutant.
Fig 5.
Neutralization activities of anti-rFSBM serum.
(A) Competitive inhibition of fibronectin binding by anti-rFSBM serum. Sera from rFSBM-immunized mice or CFA adjuvant-only control mice were incubated with emm1-type GAS for 1 h and then added to rhodamine-conjugated fibronectin for another 1 h. The MFI of the GAS was measured using a flow cytometer. (B) The rFSBM-immunized serum or the CFA adjuvant-only control serum was incubated with emm1-type GAS and then used to infect A549 or HMEC-1 cells. The unbound bacteria were removed by washing with sterile PBS and the total number of bacteria bound with the cells was quantified by plating on THY agar plates. *P< 0.05, **P< 0.01, compared with the values determined for the CFA adjuvant-only control serum. (C) The anti-rFSBM serum or CFA adjuvant-only control serum was incubated with streptolysin S and mouse RBC for 1 h, and the percentage of hemolysis was measured using an ELISA reader, as described in the Materials and Methods. *P< 0.05, compared with the values determined for the control serum.
Fig 6.
Immunization with recombinant FSBM protein protected mice against different emm type GAS-induced tissue damage and mortality.
The severity of the skin lesions (A) and the mortality (B) in the rFSBM-immunized mice were significantly decreased compared to the control mice after air pouch inoculation with 2 x 108 CFU of different emm types of GAS (A) or intraperitoneal infection with 1 x 108 CFU of different emm types of GAS (B). The survival curves were compared for significance using the log-rank test for the immunized mice versus the control group (P< 0.01).