Natural Mutations In Streptococcus Agalactiae Resulting In Abrogation Of Beta Antigen Production

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for beta antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of Streptococcus agalactiae In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed beta antigen, while five strains did not express beta antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of beta antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of beta antigen indicating the crucial regulatory role of TCS BgrRS The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of Streptococcus agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of beta antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of beta antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of Streptococcus agalactiae gives a novel clue for understanding of Streptococcus agalactiae adaptation to the human.