PROJECT SUMMARY Pregnant and post-partum women have a 20-fold increase in the incidence of invasive group A Streptococcus (GAS) infections compared with non-pregnant women, with this mostly being attributable to infections that originate in the reproductive tract ante- or post-partum (aka puerperal infections). Decades of epidemiological data have identified that serotype M28 GAS isolates, for undefined reasons, are non-randomly associated with puerperal infections. The goal of the proposed research is to characterize the function and transfer of a 36.3 kb genomic island, termed the ‘region of difference 2’ (RD2), with regard to the enhanced ability of serotype M28 GAS isolates to cause puerperal infections. This research will be of interest to the infectious diseases community because of the following observations. First, RD2 is distributed in the GAS population along serotype-specific lines, being present in all serotype M28 GAS strains but absent from most other serotypes. Second, sequence analysis of RD2 is consistent with this element being horizontally transferred into GAS from group B Streptococci (GBS), an important finding given that GBS are a common constituent of the vaginal microflora. Third, RD2-like elements are also present in other pathogens (e.g. group C and G Streptococci), expanding the relevance of the insights gained. Specific Aim 1: Assess the role of the GAS hyaluronic acid capsule in the function and transfer of the RD2 element. We have identified that the RD2 element enhances the ability of serotype M28 GAS to adhere to human vaginal epithelial cell lines and to colonize the female reproductive tract. Given the phenotype- altering consequences of harboring RD2 we believe it prudent to assess factors responsible for the transfer and function of this element in the GAS population. We will test the hypothesis that, by masking the GAS cell surface, the hyaluronic acid capsule inhibits both the cell-to-cell transfer and phenotype-altering activity of RD2. This hypothesis is supported by the recent finding that serotype M28 isolates are acapsular. Specific Aim 2: Determine whether RD2 promotes invasive GAS disease and identify the molecular basis behind the RD2-mediated remodeling of transcript levels. In the first part of this aim, we will test the hypothesis that the RD2 element promotes invasive GAS disease in addition to promoting host colonization. In the second part of this aim, we will test the hypothesis that the activity of RD2-encoded transcriptional and post-transcriptional regulators are behind our finding that transcripts from 108 core chromosomal genes (i.e. genes located outside of RD2) are altered in their abundance in the presence of RD2. Completion of this proposal will advance our understanding of strain emergence and phenotypic heterogeneity in a prevalent Gram-positive pathogen. Specifically, the data would inform on the intimate relationship between RD2 and GAS isolates, providing a link between GAS gene c...