Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci

Abstract

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.

FIG. 1.

Nucleotide sequence and deduced amino acid sequence of the gene encoding the BPS protein of GBS (EMBL accession number: AJ133114). Putative −10 and −35 sequences are boxed, and the Shine-Dalgarno region is underlined. Within the BPS coding region, the initial codons of the two repeats are boxed and the regions coding for the LPXTG membrane anchor consensus sequence and the charged C-terminal tail are underlined. Amino acids are given in one-letter code.

FIG. 2.

(A) Recombinant expression of GST-tagged BPS in E. coli XL 1-Blue MRF" and purification by glutathione-agarose affinity chromatography. The Coomassie-stained gel shows E. coli whole-cell extracts prior to induction (lane 1) and after induction (lane 2) and purified recombinant BPS fusion protein (lane 3). (B) Western blot analysis of whole-cell lysates of GBS strain Compton R (lane 1), E. coli XL 1-Blue MRF" harboring plasmid pGEX2T (lane 2), and E. coli XL 1-Blue MRF" harboring plasmid pSE4 (lane 3) using a polyclonal antiserum raised against purified recombinant GST-BPS fusion protein. (C) Western blot analysis of purified recombinant BPS protein as well as whole-cell extracts of GBS and E. coli using BPS antiserum. Lane 1, purified recombinant BPS; lane 2, Compton R; lane 3, 71-735 (serotype III/R1); lane 4, H4A-0126 (type Ia/R1, BPS); lane 5, 76-043 (type III/R4); lane 6, E. coli XL1-Blue expressing BPS; lane 7, E. coli XL1-Blue control.

FIG. 3.

Immunoelectron microscopy of S. agalactiae Compton R using a monospecific BPS antiserum. Preembedment labeling studies (A and C) clearly demonstrate that BPS protein is located at the bacterial surface. A ring of gold-particles (black dots) can be found on the outer edge of the bacterial cell wall. The control strain (76-043/III/R4), which does not express the BPS protein, exhibits only a very few gold particles (B, arrows). Postembedding (D) also reveals that BPS protein is exclusively located on the bacterial surface despite the fact that less labeling could be detected due to loss of antigenicity during fixation, dehydration, and embedding. Almost no labeling was found in the bacterial cytoplasm. Bars, 250 nm.

FIG. 4.

(A) Identification of the surface proteins of S. agalactiae Compton R by immunodiffusion in agarose slides. Center well, HCl extract of the GBS Compton R strain; well 1, antiserum to the R3, R4, and BPS proteins of Compton R; wells 2 and 5:,antiserum to the R3 and BPS proteins of Compton R; wells 3 and 6, antiserum to the recombinant BPS protein (anti-BPS); well 4, antiserum to the R3 protein from Compton R. (B) Effect of enzyme treatment (1 h at 37°C) on the immunoprecipitation reactions of the R3 and BPS proteins from the HCl extract of S. agalactiae Compton R. Center well, antiserum to the R3 and BPS proteins of Compton R; well 1, untreated HCl extract; well 2, pH 4 buffer control; well 3, extract treated with 0.2% pepsin (pH 4); well 4, extract treated with 5% trypsin (pH 8); well 5, extract treated with 0.2% trypsin; well 6, pH 8 buffer control.

FIG. 5.

Vaccination with the BPS protein triggers the elicitation of a protective response. (A) BPS-specific serum antibodies in mice as determined by ELISA after subcutaneous immunization with the BPS protein. Results are expressed as the geometric mean end point titers (GMT). Error bars, standard errors of the means.(B) Survival times of vaccinated and nonvaccinated mice after challenge with GBS. Animals were challenged with an inoculum of strain Compton R corresponding to the LD₈₀ at day 37 after the primary immunization, and mortality was recorded daily. The differences in survival observed between immunized and control animals were statistically significant (P ≤ 0.05).