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.Streptococcus agalactiae (the group B streptococcus [GBS]) possesses genotypic and phenotypic markers which are important in epidemiological settings. Among the established phenotypic markers, the capsular polysaccharides (CPSs) Ia, Ib, and II through IX play prominent roles in the classification of GBS, in the pathogenesis of GBS disease, and as targets of protective antibodies (15). A variety of surface-anchored and strain-variable proteins are also important GBS markers. These proteins make it possible to define GBS serosubtypes within each CPS type by using antibody-based or gene-based methods for subtyping (6, 11 19, 22). These proteins include Cß and the alpha-like proteins (Alps) C␣, Alp1 (formerly epsilon), Alp2 and Alp3 (formerly probably R1), and Rib (another designation for the R4 protein) (28). The Alps are characterized, among other ways, by a repeat-containing region which has a causal relation to the ladder-like patterns formed by these proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. The Alps possess chimeric sequences, show variable immunological crossreactivities, and induce increased resistance to GBS infections in experimental models (1,13,15,18,30). For this reason, th...

Novel Aspects of the Z and R3 Antigens of Streptococcus agalactiae Revealed by Immunological Testing

“…Reference and prototype strains included in the GBS strain collection of this laboratory were selected for testing. All the isolates are shown in Tables 1 and 2, and many of the isolates have been used in earlier studies (10,11). The CMFR30 strain was included in a carrier GBS strain collection from Zimbabwe (10,11).…”

“…HCl extraction of GBS whole cells was used to prepare antigen-containing extracts as described earlier (11). Briefly, bacteria were treated with 0.2 M HCl at 50°C for 2 h with frequent stirring, neutralization of the suspension by NaOH, and centrifugation, and the supernatant was precipitated with 5% (wt/vol) trichloroacetic acid and then dissolved in phosphate-buffered saline (PBS) (pH 7.2) and precipitated in 70% saturation of ammonium sulfate.…”

“…All the isolates are shown in Tables 1 and 2, and many of the isolates have been used in earlier studies (10,11). The CMFR30 strain was included in a carrier GBS strain collection from Zimbabwe (10,11). Isolates were cultured on blood agar plates or in Todd-Hewitt broth at 35°C for 20 h or for 2 days when used for preparation of whole-cell suspensions for antigen coating of enzyme-linked immunosorbent assay (ELISA) plates.…”

“…HCl-extracted materials were separated by sieve chromatography as described previously with a Sephacryl S-300 HR column (Amersham Pharmacia Biotech AB, Uppsala Sweden) and elution with PBS-NaN 3 . A DEAE Sephacel column (Amersham Pharmacia Biotech AB) was used for ion-exchange chromatography (pH 8.0) with elution by stepwise increments of NaCl from 0 to 0.5 M concentrations (11). An immunoadsorption assay was performed using a CNBractivated Sepharose 4B column (Amersham Pharmacia) according to the recommendations of the manufacturer.…”

“…These findings suggest a major role(s) of the R3 protein in GBS from certain geographical areas. In earlier experiments by some of us employing antiserum which was considered specific for the R3 protein, the R3 antiserum nevertheless contained antibodies against an additional surface-attached and strain-variable GBS protein which was named the Z antigen (11). This observation was pursued by further antibody-based experiments described in the present study, which, among other findings, showed a greater complexity than previously recognized of strain-variable antigens expressed by the R3 reference strain Prague 10/84 (ATCC 49447; serotype V/R3) and several other isolates.…”