Inactivation of DNA-binding response regulator Sak189 abrogates beta-antigen expression and affects virulence of Streptococcus agalactiae.

BACKGROUND: Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for beta-antigen, an important virulence factor. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the beta-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded beta-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. CONCLUSIONS/SIGNIFICANCE: Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bacgene regulatory system).

[Protective properties of certain external proteins of group B streptococci].

On the basis of genes, which control synthesis of externally localized proteins of group B streptococci (bac and scaAB), recombinant polypeptides P6 and ScaAB were obtained. Data on protective activity of these polypeptides during experimental infection of immunized mice as well as in opsonophagocytic test on cultivated peritoneal macrophages are presented. It has been shown that protective effect of specific antibodies to P6 was dependent from intensity of immune response. Titer of specific IgG to P6 equal 1:25000 was protective for mice during challenge with LD50. During sublethal challenge level of humoral immunity determined both rate of microorganism elimination and degree of decrease of concentration of streptococci in the spleen. Recombinant polypeptide ScaAB also had marked protective activity and protective titer ScaAB-specific IgG was significantly lower compared with the first polypeptide (1:1600). It has been established that both types of antibodies have opsonizing activity against different strains of group B streptococci. Opsonizing properties of antibodies to P6 were restricted to Bac protein-producing streptococci whereas specificity of antibodies to ScaAB was not restricted by type and group borders. Opsonization of both group B and group A streptococci was revealed. It has been established that protective efficacy mediated by antibodies was dependent not only from their opsonizing characteristics but also from availability of protein antigens, which under certain conditions can be shielded by capsular polysaccharide. It has been assumed that vaccine preparation developed on the basis of polypeptides P6 and ScaAB is promising for further research.

Protein F, a fibronectin-binding protein of Streptococcus pyogenes, also binds human fibrinogen: isolation of the protein and mapping of the binding region.

During screening of a gene library of Streptococcus pyogenes type M15 for fibrinogen-binding material, a protein of approximately 100 kDa, encoded outside the vir region, was found. DNA sequencing revealed this component to be identical to protein F, a fibronectin-binding protein. Isolation of the recombinant protein, termed F15, was performed by the use of fibrinogen affinity chromatography. The affinity constant (Ka) of protein F15 for fibrinogen, 1.25 x 10(7) mol-1, was lower than that for fibronectin, 1.8 x 10(8) mol-1. The fibrinogen-binding domain was located in the N-terminal part of the molecule, while the fibronectin-binding domains, as previously determined, were in the C-terminal portion of protein F. To examine the amino acid sequence heterogeneity of protein F, the 5' part of the prtF gene, corresponding to the N-terminal variable region of the protein, was amplified by PCR from 12 strains of S. pyogenes belonging to six different M-types. Alignment of these nucleotide sequences indicated that the 5' portion of the prtF gene had probably undergone a number of intragenic recombination and horizontal gene transfer events, allowing a pattern of structural diversity of protein F observed earlier for some other streptococcal virulence factors. There was no strict correlation between M-type and nucleotide sequence of the variable region of the prtF gene and, compared to streptococcal M protein, the overall variation observed for protein F appeared more limited.