PROJECT SUMMARY Emergence and evolution of bacterial pathogens in humans is the result of a complex interplay between bacterial factors and the host immune response. Streptococcus agalactiae (group B Streptococcus, GBS) emerged in the late 1960s and early 1970s as a frequent cause of sepsis and meningitis in infants <90 days of age. Twenty years later, GBS was also recognized as a significant cause of disease in adults. Serum cross- reactivity to the GBS capsular polysaccharide (CPS) led to the recognition of 10 CPS types (Ia/Ib, II-IX). The epidemiology of infant disease is defined by GBS strains of CPS Ia and III and much research has been devoted GBS pathogenesis and disease prevention in this age group. In contrast, adult disease is dominated by strains of CPS type IV and V of which we understand little of the bacterial mechanisms contributing to disease. Combined with the fact that of the ~30,000 cases of invasive GBS disease that occur annually in the US two-thirds occur in adults, a major knowledge gap exists in our understanding of adult GBS disease mechanisms. We previously performed one of the only studies to examine CPS V GBS and discovered that the majority (>85%) are of a single sequence type (ST), ST1, and evolve by small genetic changes (mutations). Here, we extend our previous analysis to include CPS V GBS strains isolated at the beginning of the rise in adult GBS disease. Combined with our previous analysis, our data indicate that “early” (pre-1992) CPS V GBS disease strains were more likely to be ST2 compared to contemporary CPS V GBS among which ST1 predominates. In addition, we have identified genes encoding an unstudied two-component gene regulatory system (TCS) as highly polymorphic within the ST1 population consistent with a critical role in CPS V GBS pathogenesis. These data form the basis of this proposal which seeks to test the hypothesis that ST1 GBS have altered host-pathogen interaction compared to non-ST1 strains owing to differences in gene content and global gene regulation. Using RNA-sequencing following exposure to human blood ex vivo and models of GBS colonization and disease, Specific Aim 1 will determine the contribution of gene regulatory differences between ST1 and non-ST1 GBS that contribute to differences in host-pathogen interaction and dominance of ST1 among contemporary CPS V GBS. In Specific Aim 2, we test the hypothesis that the identified TCS contributes to ST1 phenotypic characteristics and predominance among CPS V GBS. We will generate isogenic deletion mutants and compare transcriptomes to the parental strains using RNA-sequencing to define the key regulatory targets of the TCS. Subsequently, we will correlate differences in gene regulation to differences in host-pathogen interaction using models of GBS colonization and disease. Completion of these studies will provide critical new information regarding CPS V GBS disease in adults and serve as a model for investigation of other CPS types. Moreover...