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

Abstract

Group B streptococci (GBS) are important human and bovine pathogens which can be classified by a variety of phenotype-and gene-based techniques. The capsular polysaccharide and strain-variable, surface-anchored proteins are particularly important phenotypic markers. In an earlier study, a previously unrecognized protein antigen called Z was described. It was expressed by 27.2% of GBS strains from Zimbabwe, usually in combination with R3 protein expression. In this study, a putative Z-specific antiserum actually contained antibodies against two different antigens named Z1 and Z2; Z1 was >250 kDa in molecular mass. Z1, Z2, and R3 generated multiple stained bands on Western blots and showed similar chromatographic characteristics with respect to molecular mass, aggregate formation, and charge. Of 28 reference and prototype GBS strains examined, 8/28 (28.5%) isolates expressed one, two, or all three of the Z1, Z2, and R3 antigens; 4/28 expressed all three antigens; 2/28 expressed Z2 and R3; 1/28 expressed Z1 only; and 1/28 expressed R3 only. Twenty (71.5%) of the 28 isolates expressed none of the three antigens. Expression of one or more of these antigens was shown by isolates of the capsular polysaccharide types Ia, Ib, V, and IX and NT strains and occurred in combination with expression of various other strain-variable and surface-localized protein antigens. When used as serosubtype markers, Z1, Z2, and R3 affected existing GBS serotype designations for some of the isolates. For instance, the R3 reference strain Prague 10/84 (ATCC 49447) changed serotype markers from V/R3 to V/R3, Z1, and Z2. Other isolates may change correspondingly, implying consequences for GBS serotyping and research. S ubspecies classification of Streptococcus agalactiae (group B streptococci [GBS]) is important in epidemiological settingsand in efforts to identify highly virulent variants of the bacterium, which is an important pathogen in humans, notably in neonates. A variety of typing techniques have been used for this purpose, such as phenotypic marker-based and/or gene-based techniques. For instance, by use of antibody-based and molecular methods such as multilocus sequence typing (MLST) (1) or restriction digest pattern (RDP) typing (2), GBS clones with particularly high virulence for neonates have been identified (1, 2). The highly virulent variants have been associated with a gene called srr-2 and expression of a surface-linked marker, the ε antigen, which may contribute to the increased virulence (3). Other gene elements and gene products also characterize the highly virulent GBS isolates (4, 5). In addition to a core genome shared by all GBS, GBS genomes contain a large number of strain-variable genes which may encode virulence factors such as strain-variable and surface-linked proteins which may function as adhesins and/or invasins, as targets of protective antibodies (meaning potential vaccine candidates), as enzymes, and as markers for serosubtyping of GBS already serotyped on the basis of the capsular polysaccharide (CPS) antig...

The Streptococcus agalactiae R3 surface protein is encoded by sar5

“…The R4 protein has been shown to be identical to Rib and is encoded by the rib gene (15), while R5 has been renamed group B protective surface protein (BPS) and was shown to be the gene product of sar5 (13, 16). The R3 protein has been characterized to some extent (12, 17–19), and has proved useful as a serosubtype GBS marker (20, 21). However, the gene encoding the R3 protein is still unknown (Table 1).…”

The Streptococcus agalactiae R3 surface protein is encoded by sar5

“…The R4 protein has been shown to be identical to Rib and is encoded by the rib gene [17], while R5 has been renamed group B protective surface protein (BPS) and was shown to be the gene product of sar5 [15,18]. The R3 protein has been characterized to some extent [14,[19][20][21], and has proved useful as a serosubtype GBS marker [22,23]. However, the gene encoding the R3 protein is still unknown (Table 1).…”

Survey of Immunological Features of the Alpha-Like Proteins of Streptococcus agalactiae

“…This protein generated patterns with multiple bands on Western blotting whether probed with polyclonal antibodies or probed with monoclonal antibodies (48,49). R3 antibodies showed no cross-reactivity with any of the well-defined Alps, which would have been unusual if R3 had belonged to the Alp family (49). This protein may belong to another family of streptococcal proteins, but R3 awaits full characterization and sequencing of the encoding gene.…”

“…In one study, 173 human and 52 bovine GBS strains were tested, mostly by PCR, for 18 different surface-localized markers, including the Alps; all of these markers were detected in isolates of both categories, although there were some differences in the frequency of occurrence of the markers (55). The Alps showed essentially similar CPS-Alp associations in the two strain categories and behaved similarly in immunological tests with specific antisera, without displaying results that could point to immunological differences between the Alps of human and bovine GBS, or differences between the non-Alp proteins Z1, Z2, and R3 (49,55). More detailed comparative testing, including comparative sequence analysis, may be required to determine whether surface-exposed protein markers of human and bovine GBS differ significantly.…”

“…R3 is a high-molecular-mass protein among others expressed by the prototype strains 10/84 (ATCC 49447) and 9828 (NCTC 9828). This protein generated patterns with multiple bands on Western blotting whether probed with polyclonal antibodies or probed with monoclonal antibodies (48,49). R3 antibodies showed no cross-reactivity with any of the well-defined Alps, which would have been unusual if R3 had belonged to the Alp family (49).…”