ABSTRACT: While Streptococcus pneumoniae (Spn) is generally thought as an extracellular pathogen, its ability to cross through vascular endothelial cells (VEC) is a critical aspect of its pathogenesis. VEC transcytosis enables Spn to escape the airway during pneumonia and cause bacteremia; it also permits Spn in the vasculature to invade the heart and central nervous system. Spn replication and cytotoxicity at these and other sites of dissemination is devastating and the consequences include life-long disability or death. Thus, gaining a better understanding of how Spn interacts with VEC is critical in order to identify novel therapeutics that can block disease progression. Clinical isolates of Spn are almost always encapsulated. One way capsule protects the bacterium from opsonophagocytosis is by impeding Spn interactions with host cells. Importantly, and despite the fact that capsule is inhibitory of initial Spn attachment to VEC, we have collected seminal data that shows capsule is subsequently required for Spn translocation across VEC; conferring resistance to the low pH environment and reactive oxygen species (ROS) found within endosomes. Notably, isogenic mutants carrying distinct capsule types differed in their rate of successful translocation after invasion, resistance to noxious agents, and the ability to invade organs from the bloodstream. Thus, the biochemical properties of each capsule potentially explains why different serotypes of Spn have distinct propensity for invasive disease. Protection against intracellular killing / permissiveness of VEC translocation is a new virulence mechanism for capsular polysaccharide. Our hypothesis is that capsule potentiates Spn tissue invasion from the bloodstream by modulating intracellular fate. Testing of this hypothesis will inform us on the molecular basis of pneumococcal pathogenesis and may lead to the identification of novel intervention targets. Because many other bacterial pathogens produce a capsular polysaccharide, results from these studies may have broad applicability to our general understanding of host-pathogens interactions. To test our hypothesis we will: AIM 1. Determine how capsule impacts interactions with VEC. 1.a: Categorize Spn capsule types as permissive versus non-permissive for VEC transcytosis. 1.b: Determine the importance of capsule shedding during VEC transcytosis. 1.c: Identify the biochemical properties of capsule that protect intracellular Spn. AIM 2. Determine how capsule impacts disseminated disease. 2.a: Determine how capsule impacts translocation across epithelial::endothelial cell barriers. 2.b: Determine how capsule type impacts progression from pneumonia to disseminated invasive disease.