Putative novel surface-exposed Streptococcus agalactiae protein frequently expressed by the group B streptococcus from Zimbabwe

Abstract

Group B streptococci (GBS) express a variety of surface-exposed and strain-variable proteins which function as phenotypic markers and as antigens which are able to induce protective immunity in experimental settings. Among these proteins, the chimeric and immunologically cross-reacting alpha-like proteins are particularly important. Another protein, R3, which has been less well studied, occurred at a frequency of 21.5% in GBS from Zimbabwe and, notably, occurred in serotype V strains at a frequency of 75.9%. Working with rabbit antiserum raised against the R3 reference strain ATCC 49447 (strain 10/84; serotype V/R3) to detect the expression of the R3 protein, we recorded findings which suggested that strain 10/84 expressed a strain-variable protein antigen, in addition to R3. The antigen was detected by various enzyme-linked immunosorbent assay-based tests by using acid extract antigens or GBS whole-cell coats and by whole-cell-based Western blotting. We named the putative novel antigen the Z antigen. The Z antigen was a high-molecular-mass antigen that was susceptible to degradation by pepsin and trypsin but that was resistant to m-periodate oxidation and failed to show immunological cross-reactivity with any of a variety of other GBS protein antigens. The Z antigen was expressed by 33/121 (27.2%) of strains of a Zimbabwean GBS strain collection and by 64.2% and 72.4% of the type Ib and type V strains, respectively, and was occasionally expressed by GBS of other capsular serotypes. Thus, the putative novel GBS protein named Z showed distinct capsular antigen associations and presented as an important phenotypic marker in GBS from Zimbabwe. It may be an important antigen in GBS from larger areas of southern Africa. Its prevalence in GBS from Western countries is not known.

FIG. 1.

ELISA inhibition with HCl extracts tested for inhibition of binding of Z PAb (1:2,000) to the CMFR30 (Ib/Cß) extract coat (A) and inhibition of binding of R3 PAb (1:2,000) to the 9828 (NT/Alp4, R3) extract coat (B). The HCl extracts tested for inhibition are indicated.

FIG. 2.

Double-diffusion testing of HCl extract of GBS strain GMFV223T (Ib/Cß, Cα) (well 1), strain CMFR30 (Ib/Cß) (well 2), strain 10/84 (V/R3) (well 3), and strain 9828 (NT/Alp4, R3) (well 4) tested against the original R3 PAb, i.e., serum with both anti-R3 and anti-Z antibodies (well 5) diluted 1:2. For the method used to prepare the original R3 PAb, see the text and footnote a of Table 1.

FIG. 3.

(A) Fractionation of material in an HCl extract of strain 10/84 (ATCC 49447; V/R3) on a Sephacryl S-300 HR column. Fractions were monitored at 280 nm and tested (1:5) by ELISA for recognition by Z PAb (1:1,000) and R3 PAb (1:2,000). The fractions corresponding to the void volume (V₀) and the total volume (V_t) and those with the peak ability to coat human IgM and IgG in the ELISA are indicated. (B) Materials in fractions 32 to 40 from the Sephacryl S-300 HR column applied to a DEAE Sephacel column and eluted with stepwise increments of NaCl concentrations in basic buffer. The activities of the eluted components (1:5) as the coating antigen for the binding of Z PAb and R3 PAb are shown.

FIG. 4.

Western blot of SDS lysates of whole cells of GBS strains. Lysates from strain 10/84 (strain ATCC 49447; V/R3) (lane a), strain 9828 (NT/Alp4, R3) (lane b), and strain CMFR30 (V/Cß) (lane c) were probed with Z PAb (1:1,000) (A) and R3 PAb (1:1,000) (B). (C) Lysates from strain 10/84 (lane 1), four Z-antigen-positive Zimbabwean GBS strains (lanes 2, 4, 6, and 8), and four Z-antigen-negative strains (lanes 3, 5, 7, and 9) were probed with Z PAb (1:1,000). Protein standards are shown to the left.