Deletion of repeats in the alpha C protein enhances the pathogenicity of group B streptococci in immune mice

Abstract

The alpha C protein is a protective surface-associated antigen of group B streptococci (GBS). The prototype alpha C protein of GBS (strain A909) contains nine identical tandem repeats, each comprising 82 amino acids, flanked by N- and C-terminal domains. Clinical isolates of GBS show variable numbers of repeats with a normal distribution and a median of 9 to 10 repeats. Here, we show that escape mutants of GBS expressing one-repeat alpha C protein were 100-fold more pathogenic than GBS expressing wild-type nine-repeat alpha C protein in neonatal mice whose dams were immunized with antiserum elicited to nine-repeat alpha C protein (50% lethal doses of 1.6 x 10(3) and 1.8 x 10(5), respectively; P = 0.0073). There was no difference in pathogenicity in nonimmune mice. Enzyme-linked immunosorbent assay inhibition showed that nine-repeat but not one-repeat alpha C protein is readily available for antibody binding on the surface of intact GBS. Immune electron microscopy studies with antibodies to the capsular polysaccharide (CPS) and to the alpha C protein demonstrated localization of the nine-repeat alpha C protein and the CPS at similar distances from the cell wall. The one-repeat alpha C protein was visualized poorly and only in close proximity to the cell wall, thus suggesting that antibody binding to the protein was hindered by CPS or other cell surface components. We concluded that deletion in the repeat region of the alpha C protein enhanced the pathogenicity of GBS in immune mice by (i) loss of a protective (conformational) epitope(s) and (ii) loss of antibody binding to the alpha C protein due to a decrease in antigen size relative to cell wall components and/or CPS.

FIG. 1

ELISA inhibition with purified alpha C protein with different repeat numbers (1, 2, 9, and 16 repeats [rep]) as inhibiting antigen (Inh Ag) (left) and intact GBS with different repeat numbers (1, 2, 9, and 18 repeats) as inhibiting antigen (right). In both experiments rabbit antiserum to nine-repeat alpha C protein (final dilution, 1:8,000) was used. Microtiter plates were coated with purified nine-repeat alpha C protein at 0.125 μg/ml. Twofold dilutions were made from the inhibiting antigen (10-μg/ml starting dilution of purified alpha C protein; 10⁹-cell/ml starting dilution of intact GBS). The left panel has been modified from reference and is shown here for comparison.

FIG. 2

ELISA inhibition with purified alpha C protein with different repeat numbers (1, 2, 9, and 16 repeats [rep]) as inhibiting antigen (Inh Ag) (left) and intact GBS with different repeat numbers (1, 2, 9, and 18 repeats) as inhibiting antigen (right). In both experiments rabbit antiserum to one-repeat alpha C protein was used (final dilution, 1:8,000). Microtiter plates were coated with purified one-repeat alpha C protein at 1 μg/ml. Twofold dilutions were made from the inhibiting antigen (10-μg/ml starting dilution of purified alpha C protein; 10⁹-cell/ml starting dilution of intact GBS).

FIG. 3

EM photographs of GBS with one-repeat alpha C protein (A and C) and nine-repeat alpha C protein (B and D) at the cell surface, incubated with one-repeat alpha C protein-specific rabbit antiserum (A and B) or with rabbit antiserum to CPS type Ia (C and D). For protein staining, 15-nm-diameter-gold-labelled protein A was used, and for the CPS type Ia staining, 20-nm-diameter-gold-labelled protein A was used (final dilution, 1:50). Bar, 500 nm.

FIG. 4

Distribution of repeat number in the alpha C proteins from escape mutants of GBS isolated from the spleens of mice immunized with one-repeat alpha C protein antiserum (left) or nine-repeat alpha C protein antiserum (right). Repeat numbers were determined by Western blotting with monoclonal antibody 4G8 or rabbit antiserum to nine-repeat alpha C protein.

FIG. 5

Western blots of escape mutants of GBS isolated from the spleens of pups immunized with antiserum to one-repeat alpha C protein (A) or to nine-repeat alpha C protein (B). These blots were incubated with antiserum to nine-repeat alpha C protein. (A) Wild-type GBS with nine-repeat alpha C protein (lane 1) and escape mutants with nine-repeat alpha C protein (lanes 2 to 14); (B) wild-type GBS with nine repeats (lane 1) and escape mutants with zero repeats (lane 9), with two repeats (lanes 11 to 14), with five repeats (lanes 2, 4, and 8), and with nine repeats (lanes 3, 5, 6, 7, and 10). Numbers on the left are molecular masses in kilodaltons.

FIG. 6

Distribution of repeat number in the alpha C protein in clinical isolates of GBS. Repeat numbers were determined by Western blotting with 4G8, a repeat-specific monoclonal antibody. This figure was derived from data in a previous study (16).