PROJECT SUMMARY Group B Streptococcus (GBS) remains the leading etiologic agent of neonatal bacterial meningitis and a major opportunistic pathogen in certain adult populations, including pregnant women. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20-30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal meningitis upon GBS disruption of the blood-brain barrier (BBB) and 10-15% mortality, regardless of antibiotic treatment. Despite this major public health concern, the specific mechanisms and regulation of Group B streptococcal effectors that mediate successful colonization and development of meningitis remain unknown. Recently, Cas9, an endonuclease traditionally known for its role in CRISPR/Cas bacterial adaptive immunity, was shown to attenuate virulence and the interaction of pathogens with host cells through endogenous gene regulation. However, the role of Cas9 in the colonization and pathogenesis of other pathogens, such as GBS, has not been well-studied. My preliminary work shows that GBS cas9 mutants are attenuated in vivo using models of GBS vaginal colonization and hematogenous meningitis and invade brain endothelium and vaginal epithelium significantly less than WT GBS, in vitro. Further, RNA-Seq analysis indicated that Cas9 globally regulates gene expression in GBS, including dysregulation of putative virulence factors, metabolic and cell wall formation factors, and riboflavin synthesis machinery. Based on my preliminary data, this proposal seeks to elucidate the molecular mechanisms by which Cas9 globally regulates GBS factors, such as the riboflavin pathway, and to determine how dysregulated riboflavins might affect GBS evasion of host immunity during vaginal colonization. These questions will be addressed with both in vitro and in vivo models of GBS vaginal colonization and BBB penetration in the following specific aims: AIM 1: Identify components of the CRISPR/cas operon and Cas9 residues that contribute to Cas9-mediated gene regulation of GBS factors; AIM 2: Determine if Cas9 regulates global GBS gene expression at the genomic or transcriptomic level; AIM 3: Characterize the role of Cas9-regulated riboflavin synthesis in GBS immune evasion. This proposal is the first to examine both the mechanism of Cas9- regulation of GBS factors and the role of these factors in host immune response to GBS, which may ultimately afford novel targets and alternative therapeutic strategies.