The Streptococcus agalactiae R3 surface protein is encoded by sar5

Streptococcus agalactiae (group B streptococcus; GBS) is an important human pathogen causing pneumonia, sepsis and meningitis in neonates, as well as infections in pregnant women, immunocompromised individuals, and the elderly. For the future control of GBS-inflicted disease, GBS surface exposed proteins are particularly relevant as they may act as antigens for vaccine development and/or as serosubtype markers in epidemiological settings. Even so, the genes encoding some of the surface proteins established as serosubtype markers by antibody-based methods, like the R3 surface protein, are still unknown. Here, by examining a Norwegian GBS collection consisting of 140 strains, we find that R3 protein expression correlates with the presence of the gene sar5. By inducible expression of sar5 in an R3-negative bacterial strain we show that the sar5 gene product is specifically recognized by an R3 monoclonal antibody. With this we identify sar5 as the gene encoding the R3 surface protein, a serosubtype marker of hitherto unknown genetic origin.

with specific serotypes, and thus used to define serosubtypes (6). Historically, detection of 72 serosubtypes by means of antibody-based methods has played a major role. In more recent 73 years, serosubtyping of GBS has benefitted greatly from the introduction of molecular 74 methods, such as PCR and whole genome sequencing (WGS) (7,8).  (Table 1). However, there is still some discrepancy and confusion 78 surrounding the traditional nomenclature, and some surface proteins that have not yet been 79 definitely linked to a specific gene. One classification scheme of GBS surface proteins includes 80 C and the Cα-like proteins (Alps) Cα, Alp1-4 and Rib. Nearly all GBS strains carry one of 81 the six alp genes (Alp GBS) although, occasionally, an Alp-encoding gene may be absent (non-82 Alp GBS) (9). Another, and overlapping, classification system of GBS surface proteins is the 83 Streptococcal R proteins first described in 1952 (10), which are resistant to trypsin digestion 84 (thereby designated "R"). R proteins are categorized into five types, R1-5 (11-13). R1 is 85 probably non-existent as a distinct protein; the antiserum raised against R1 was later shown to 86 recognize the identical N-termini of Alp2 and Alp3, the gene products of alp2 and alp3, 87 respectively (14). The R2 protein is expressed by group A and C streptococci and does not 88 seem to occur in GBS (13). The R4 protein has been shown to be identical to Rib and is encoded 89 by the rib gene (15), while R5 has been renamed group B protective surface protein (BPS) and 90 was shown to be the gene product of sar5 (13, 16). The R3 protein has been characterized to 91 some extent (12,(17)(18)(19), and has proved useful as a serosubtype GBS marker (20, 21).

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However, the gene encoding the R3 protein is still unknown (Table 1). BPS was initially 93 thought to be distinct from R3 (13), however, a later study pinpointed a correlation between 94 the presence of the BPS-encoding sar5 gene and R3 expression (6). Here, we follow up on this 95 correlation, hypothesizing that sar5 encodes R3. Unraveling the R3-encoding gene, and the 96 putative discrepancy in the nomenclature and nature of the sar5 gene product, is important for 97 the sar5 gene product as a prospective target in vaccine development and molecular based GBS 98 serosubtyping, as well as for functional studies on its mechanistic role in pathogenicity. The sar5 positive R3 negative GBS strains express R3 but encode a sar5 deletion variant.

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Thus, these strains were in conflict with our hypothesis that sar5 encodes R3. The initial  The sar5-encoded protein is recognized by the R3 antibody. 168 Based on the above results, we had strong indications that the sar5 gene encodes the R3 protein. 169 We aimed to prove this experimentally by inducing sar5 protein expression in a sar5 negative 170 bacterial species, followed by R3 protein detection. First, we constructed a sar5 inducible 171 expression vector by replacing the luciferase reporter gene of pKT1 (30)  both the R3 and the FLAG antibody bound to the induced sar5D gene product (Figure 4).

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Compared to the full-length sar5, the sar5D gene encoded a seemingly truncated R3 protein, corresponding in size to the R3 protein expressed by the 93-33 and 94-3 strains. Taken together, 192 our results demonstrate that sar5 encodes a protein recognized by the R3-specific antibody. shown in S1   invasive GBS strains expressing BPS found no overlap between R3-and BPS-expression (34).

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The presumed R3-specific antibody used in that study was also prepared by adsorbing antisera 324 made by immunizing a rabbit with GBS strain NCTC 9828. Regarding BPS and R3 as two 325 distinct proteins would result in an R3-designated antibody without antibodies targeting the 326 sar5 gene product. Using NCTC 9828 as a reference strain for R3 would thus result in R3 327 antiserum that may actually detect Alp4.

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As a consequence of our findings, already reported data on BPS and R3 are equally relevant 330 for the sar5 gene product. Using BPS as the future designation of the sar5 gene product makes 331 the historical R3 protein nonexistent, and vice versa. The nomenclature of GBS surface proteins 332 is already confusing (Table 1)