PROJECT SUMMARY Streptococcus agalactiae (Group B Strep, GBS) infections in neonates are often fatal and are strongly associated with maternal GBS vaginal colonization. The use of preventive intrapartum antibiotics, while effective against early onset disease, have their own pitfalls and have no effect on late onset neonatal GBS diseases. As such, a more long-term strategy is required to control infections by GBS. This proposal focuses on a novel GBS protein, Sak_1753, and we aim to determine its role in GBS vaginal colonization and its eligibility as a protein-based vaccine candidate. sak_1753 was previously identified as the most highly upregulated gene (~3000 fold) in the GBS A909 transcriptome when comparing vaginal colonization to growth in liquid culture. Sak_1753 is regulated directly by a two-component system SaeRS and has no known homologs outside streptococci or predicted protein domains, leaving its function a mystery. The protein is conserved among all sequenced strains of GBS and is made up of multiple repeat domains with the number of repeats differing between strains. We will characterize Sak_1753 regulation and localization and determine its role in vaginal colonization. We have created knockout (KO; ?sak_1753::Spec) and over-expression (OE; PrecA-sak_1753) strains in a wildtype (WT) GBS A909 background. Preliminary adherence assays with human vaginal epithelial cells (VK2) demonstrated that the OE strains were able to bind to epithelial cells significantly more than WT and the KO bound significantly less. Sak_1753 also showed binding specificity to collagen 1 and fibrinogen. In addition, the sak_1753 KO strain exhibited significant decreased vaginal colonization in a murine model. Initial immunofluorescence microscopy indicates that Sak_1753 is found on the cell surface, indicating a possible adhesive role of this protein. In this proposal we will further characterize the role of Sak_1753. GBS strains will be created to contain truncations of Sak_1753 in an effort to identify the role of the repeated domains in host attachment. Strains expressing truncated proteins will be tested in adhesion assays with VK2 cells and in vivo murine colonization. To further ascertain the cellular localization of Sak_1753 western blot techniques and immunofluorescence microscopy will be used. The success of this research will contribute to the strategies for reduction of GBS adhesion to vaginal tissue and hence a reduction in GBS-related neonatal diseases and deaths.