PROJECT SUMMARY Group B Streptococcus (GBS, also known as Streptococcus agalactiae) is a Gram-positive, -hemolytic bacterium normally found in the human gastrointestinal and urogenital tracts. GBS remains a leading cause of invasive disease in newborns and certain adult populations including pregnant women. The development of GBS disease is initiated by the asymptomatic colonization of the female genital tract and during pregnancy can be associated with chorioamnionitis, puerperal endometritis and preterm labor. Approximately 20-30% of healthy women are colonized rectovaginally with GBS; the majority of infants born to these women will themselves become colonized with the bacterium. Newborn infection also results from ascending infection of the bacterium through the placental membranes to initiate infection in utero. GBS is the principle etiologic agent of neonatal bacterial meningitis, and GBS meningeal infection results in 10-15% mortality despite antibiotic treatment. Additionally, up to 40% of survivors develop permanent neurological sequelae, including cognitive defects, seizure activity, and cerebral ischemia. To access the central nervous system (CNS) and cause meningitis blood-borne GBS must penetrate the blood-brain barrier (BBB); however, little is known about the very first and crucial interaction between GBS and the BBB that initiates bacterial crossing and disease progression. This proposal seeks to characterize the role of a newly identified GBS Antigen I/II family adhesin, BspC, to the pathogenesis of colonization and CNS disease. We have recently determined that BspC promotes attachment to BBB endothelium and contributes to the development of GBS meningitis. Further we have discovered that BspC interacts directly with vimentin, a widely distributed intermediate filament protein found in blood vessel endothelial cells. Thisproposal seeks toidentify the region on BspC that interacts with vimentin, and to characterize the role of this interaction in GBS meningitis and colonization of the female reproductive tract. I hypothesize that the binding region is contained within the globular variable domain (V- domain) of BspC and contributes to both GBS meningitis and vaginal colonization/ascending infection. These hypotheses will be addressed with both in vitro and in vivo models of BBB penetration and vaginal colonization in the following specific aims: Aim 1: Identify the critical regions and amino acids required for BspC V-domain- vimentin interaction; Aim 2: Characterize the role of BspC and vimentin in the female reproductive tract. These data will clarify the crucial role of Antigen I/II proteins to GBS colonization and disease and provide fundamental mechanistic insights that may inform new treatment strategies to prevent colonization and ultimately bacterial meningitis.