Characterization of a novel leucine-rich repeat protein antigen from group B streptococci that elicits protective immunity

Abstract

Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.

FIG. 1.

Alignment of the amino acid sequence of the LrrG protein from S. agalactiae (97/0099) with LRR proteins from S. pyogenes (GAS; hypothetical protein), B. forsythus (surface antigen BspA), and S. pneumoniae (PcpA). SP, signal peptide region; N, amino terminus; pr, partial repeat; C, carboxyl terminus; LPxTG, LPXTG cell wall anchoring motif. The individual repeats for LrrG are numbered. Numerals below the maps indicate the number of amino acid residues in each protein domain.

FIG. 2.

Alignment of the 22-amino-acid repeats of the LrrG protein and the consensus LRR protein motif. The amino acid positions of the repeats are indicated on the left. Amino acid residues are indicated in the sequences by the one-letter code and are included if present at a particular position in more than half of the repeats. Amino acid positions with identical or similar amino acid substitutions within the LrrG repeat region and LRR consensus sequences are highlighted.

FIG. 3.

Southern blot hybridization of chromosomal DNAs from various streptococcal species with the lrrG gene. Lanes (left to right): Ia, S. agalactiae 515a and S. agalactiae A909; Ib, S. agalactiae SB35 and S. agalactiae H36B; II, S. agalactiae 18RS21, S. agalactiae 1954/92, S. agalactiae 118/158, and S. agalactiae BS29; III, S. agalactiae BM110, S. agalactiae BS30, S. agalactiae 97/0099, and S. agalactiae M781; IV, S. agalactiae 3139; V, S. agalactiae 1169-NT; VI, S. agalactiae GBS6; VII, S. agalactiae 7271; VIII, S. agalactiae JM9; S.pyg, S. pyogenes M1; S.pneu, S. pneumoniae VH14; M, 1-kb molecular weight markers. The sizes of the chromosomal DNA fragments hybridizing with the lrrG gene are indicated on the left. Hybridization with the DIG-labeled probe was carried out at 42°C.

FIG. 4.

Surface localization of LrrG. (A) Kyte-Doolittle plot of the deduced amino acid sequence encoded by lrrG of GBS. Hydrophobic regions are above the horizontal line; hydrophobicity is indicated on the vertical axis. Regions representing a putative secretion signal peptide (SP) and an LPXTG motif are indicated. (B) Western blot of cell protein fractions from mutanolysin-treated GBS strain 97/0099 (type III) probed with anti-LrrG antisera. Lanes: 1, cell cytosol; 2, cell membrane; 3, cell wall; 4, supernatant; 5, purified recombinant LrrG stained with Coomassie blue. Molecular mass standards are indicated on the left.

FIG. 5.

Serum ELISA titers of antigen (Ag)-specific total IgG (A) and antigen-specific IgG1 and IgG2a (B) antibodies following immunization with the indicated proteins. Groups of 6-week-old CBA/Ca mice were vaccinated s.c. at days 0 and 28 with 25 μg of the respective protein vaccine mixed in a 1:1 ratio with alum adjuvant. Mice were bled from the tail at days (d) 21 and 42 before bacterial challenge on day 56. Sera were analyzed by ELISAs, and the end-point titers were determined. The values represent the mean ± standard error of the mean for 12 mice.

FIG. 6.

Vaccination with purified LrrG confers protection from challenge with GBS. Groups of 12 CBA/Ca mice aged between 6 and 7 weeks were immunized s.c. with 25 μg of protein antigen in a 1:1 mixture with alum adjuvant (final volume, 200 μl). Mice were immunized on day 0 and boosted on day 28. Mice were challenged intraperitoneally 21 days later with a lethal dose of GBS (3.5 × 10⁶ CFU). Deaths were recorded hourly, and the percent survival was determined for up to 7 days following GBS challenge. The final ratios (number of surviving mice/number of mice challenged) are indicated for groups immunized with rRib and recombinant LrrG. Statistically significant differences (P < 0.01) in survival between experimental groups and the control group (BSA-alum-vaccinated mice) are indicated by asterisks and were calculated by using the Mann-Whitney U test. Symbols: ◊, BSA-alum; □, rRib-alum; ○, LrrG-alum.

FIG. 7.

LrrG binding to epithelial cells. LrrG and truncated versions of the antigen (A) were purified by affinity chromatography and incubated in a dose-dependent manner with ME180 cells (B) and HEp-2 cells (C) for 2 h at 37°C. Bound recombinant proteins was detected with antibody specific to the His tag present on each of the recombinant proteins followed by alkaline phosphatase-coupled anti-mouse IgG. The inclusion of the AroD protein served as a negative control and to record nonspecific protein-host cell interactions. Binding experiments were carried out in triplicate, and data represent the mean ± standard error of the mean. Numerals below the maps indicate the number of amino acid (aa) residues in each region. SP, signal peptide; O.D 405nm, optical density at 405 nm.