The putative R1 protein of <i>Streptococcus agalactiae</i> as serotype marker and target of protective antibodies

Moyo, Sylvester R.; Mæland, Johan A.; Lyng, Randi Valsø

doi:10.1034/j.1600-0463.2001.091206.x

Cited by 6 publications

(15 citation statements)

References 18 publications

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“…Harvested bacteria of the GBS strains ATCC 12403 (III/R1 alp2) and 64/95 (V/R1 alp3), both of which were rib negative by PCR, and the alp-negative strains 335 (Ia/C␣) and 65604 (III/R4 rib) were extracted with trichloroacetic acid, and the material was applied to a Sephacryl S-200 HR column. The fractions were tested for the presence of Alp2 or Alp3 by probing with an anti-R1 protein serum (20), which could be done due to the cross-reactivity of R1 antibodies with Alp (12,13), and for the presence of C␣ or R4 by probing with the appropriate monoclonal or polyclonal antibodies (1, 2). All four proteins were separated from vast amounts of constituent material that absorbed UV light as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…GBS strains used in this study were reference, prototype, and clinical isolates described in previous reports (10,20). For the preparation of the protein antigens C␣, Alp2, Alp3, and R4, the following GBS strains were used: NCTC 12906 (strain 335, serotype Ia/C␣), which is negative for the genes alp and rib; strain ATCC 12403 (D136C) (serotype III/R1 alp2), which has been subjected to complete genome sequencing and is negative for bca, alp3, and rib (7); the clinical strain 64/95 (serotype V/R1 alp3), which is alp2 and rib negative; and strain 65604 (serotype III/R4 rib), which is alp negative.…”

Section: Methodsmentioning

confidence: 99%

“…Murine monoclonal antibodies against C␣ (1) and R4 (2) were used in enzyme-linked immunosorbent assays (ELISA) to identify these proteins in gel filtration fractions. A rabbit anti-R1 protein serum raised against the alp2-positive strain ATCC 12403 (20) was used for identification of Alp.…”

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Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

Mæland

Bevanger

Lyng

2004

Clin Vaccine Immunol

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The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (C␣) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological crossreactivity with C␣. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISAbased results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. Streptococcus agalactiae (group B streptococcus [GBS]) has been classified into nine serotypes based on the capsular polysaccharide antigens Ia, Ib, and II through VIII. In addition, several surface-anchored protein antigens that vary by strain have been described for GBS, and these antigens can be used to define serosubtypes (11,18). The proteins include the immunoglobulin A-binding beta C protein (C␤) (4) encoded by bac (8), the alpha C protein (C␣) (4) encoded by bca (16), and the proteins R1, R3, and R4 (5, 6, 14). C␣ and the R proteins belong to a family of so-called ladder-forming proteins (25), a designation based on the banding patterns generated on Western blotting. More recently, other laddering GBS proteins, including protein Rib (24) encoded by rib (25) and the alphalike proteins 2 and 3 (Alp2 and Alp3) (12, 13), which are gene products of alp2 and alp3, respectively (12), have been described. The relationship between the recently described proteins and the classic R proteins has been uncertain, but evidence that the proteins Rib and R4 may be identical (2; B. Smith, A. Flores, J. Dechaine, J. Krepela, A. Bergdall, and P. Ferrieri, Abstr. 15th Lancefield Int. Symp. Streptococci Streptococcal Dis., abstr. P4. 07,2002) and that Alp2 and Alp3 may be variants of the R1 protein (J. Maeland and R. V. Lyng, Abstr. 13th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P611, 2003) has been provided.Stretches of the ladder-forming proteins show sequence homology ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

Mæland

Bevanger

Lyng

2004

Clin Vaccine Immunol

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“…The GBS reference and prototype strains used in this study were those listed in Table 2, seven additional strains which have been described in previous reports (14,26), and the type VIII strain JM9 (17). These strains included at least one isolate of each of the nine capsular antigen types of GBS, and strains which expressed at least one of the well-defined, strain-variable, and surface-localized GBS proteins.…”

Section: Methodsmentioning

confidence: 99%

“…Horizontal transfer of genetic elements between strains followed by recombinational events has been advocated as an explanation of the structural relatedness and mosaicism of these proteins (16). These proteins may be important virulence factors in GBS, and they elicit antibodies which are protective in animal models (17,21,26,31,32,33). Some of the proteins show serological cross-reactivity (17,19,31,32) attributed to structural matching, and this reactivity may hamper the reliability of antibody-based protein detection, for instance, in GBS serotyping.…”

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Immunological Markers of the R4 Protein of Streptococcus agalactiae

Mæland

Bevanger

Lyng

2005

Clin Vaccine Immunol

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This study focuses on immunological markers of R4, an important Streptococcus group B (GBS) protein. The results obtained by using rabbit antisera and purified proteins for antigens in enzyme-linked immunosorbent assay-based experiments provided evidence that R4 possesses two antigenic determinants. One of the determinants is shared with the alpha-like protein 3 (Alp3) of GBS, was named R4/Alp3 common, and was expressed by GBS, which possessed the Alp3-encoding gene alp3 or the R4-encoding gene rib. The other antigenic determinant was detected only in rib-positive GBS organisms and was named R4 specific. This determinant probably is an immunological marker unique to the R4 protein. Neither of the antigenic R4 determinants showed serological cross-reactivity with the GBS proteins C␣, C␤, and R3 or with alpha-like protein 2. Of 60 clinical serotype III GBS strains, 56 (93%) isolates possessed the rib gene and 50 (89%) of the rib-positive isolates expressed levels of R4 detectable by antibody-based tests, consistent with R4 expression failure or low-level expression in ϳ10% of rib-positive GBS. alp3 was not detected in type III GBS but was possessed by six of eight type V strains and six of six type VIII strains. All alp3-positive strains were recognized by the R4/Alp3 common antibodies, but none of them were recognized by the R4-specific antibodies. NCTC 9828, a reference strain for R3 and R4, expressed the determinant R4/Alp3 common but not R4 specific. A monoclonal R4 antibody, previously considered to be R4 specific and used in GBS serotyping, targeted R4/Alp3 common and is thus not R4 specific. The results show that failure to discriminate between R4 specific and R4/Alp3 common by antisera designed for GBS serotyping can result in the false identification of Alp3 as R4 or vice versa, whereas anti-R4 antibodies targeting only the determinant R4 specific will detect only R4. Both R4 and Alp3 need further evaluation with respect to the immunobiological function of each distinct antigenic determinant, for instance, with regard to their potential as GBS vaccine components.Streptococcus agalactiae (group B streptococci [GBS]) is an important cause of infections in humans, notably in neonates. Serotyping based on the capsular polysaccharide antigens Ia, Ib, and II through VIII has been used extensively in epidemiological classification of GBS, sometimes supplemented by serosubtyping on the basis of strain-variable and surface-anchored protein antigens. These proteins include the C proteins C␣ (3) (encoded by bca [22]) and C␤ (3) (encoded by bac [10]) and the classical R proteins R1, R3, and R4 (8, 18, 35 P611, 2003). These proteins, except for C␤, belong to a protein family characterized among other things by similarity in primary structure, with up to 100% homology for some of the protein stretches (16,34), and by their generation of ladder-like patterns on Western blots, probably due to large and identical repeat units which vary in number from strain to strain (9,22,34). Horizontal transfer of genetic elements be...

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