FIG. 3.

The capsule of S. agalactiae inhibits binding of antibodies to Blr but not to protein Rib. (A) Binding of anti-Blr antibodies to S. agalactiae strain BM110 wild type (wt) and mutants of this strain. For each bacterial strain, a series of identical samples were mixed with antisera and diluted as indicated, and bound antibodies were detected as described in Materials and Methods. The mutants used were those described in the legend to Fig. 2. (B) Binding of anti-Blr antibodies to S. agalactiae strain COH1 and its acapsular mutant, COH1-13 (Cap⁻). (C) Binding of anti-Blr antibodies to S. agalactiae wt strain BM110 and its acapsular mutant, BM110-22 (Cap⁻), analyzed by immunofluorescence. A control with preimmune (preimm) serum is shown for BM110-22. Bound antibodies were detected as described in Materials and Methods, and representative images from one experiment are presented. For each analysis, the same field is shown in the phase-contrast panel (Phase) and in the immunofluorescence panel (IF). (D) Western blot analysis of bacterial extracts, using anti-Blr as the probe. The extracts were prepared from S. agalactiae BM110 wild type, its Rib- and capsule-negative double mutant (Rm69-16), and the Blr-negative BM110 mutant, Δblr-36. Molecular weight markers (in thousands) are noted at the left of blots. (E) Lack of capsule does not affect surface exposure of protein Rib. Anti-Rib antibodies, diluted as indicated, were incubated with BM110 wild type and its acapsular mutant, BM110-22 (Cap⁻). This panel was derived from data presented in Fig. 2A and B. The binding experiments shown in panels A, B, and E were performed three times with triplicate samples, and each panel represents data from one experiment. Binding observed with preimmune serum (<8%) has been subtracted.